Natural ligand for orphan G protein coupled receptor GPR86 and methods of use

ABSTRACT

The present invention is related to a method of detecting the dysregulation of GPR86 activity.

PRIORITY

This application is a divisional of application Ser. No. 10/308,968,filed Dec. 3, 2002 which claims priority under 35 U.S.C. §120 as acontinuation of International Application Number PCT/EP02/08761, filedAug. 6, 2002, which claims priority as a continuation in part of U.S.application Ser. No. 09/924,125, filed Aug. 7, 2001. The entireteachings of the above application(s) are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is related to the natural ligand for the orphan Gprotein coupled receptor GPR86 and methods of use.

BACKGROUND OF THE INVENTION AND STATE OF THE ART

Adenine and uridine nucleotides induce pharmacological and physiologicalresponses through several G-protein-coupled receptors (P2Y) andligand-gated cation channels (P2X) (1, 2). The P2Y family encompassestwo selective purinoceptors: the human P2Y₁ and P2Y₁₁ receptors whichare preferentially activated respectively by ADP and ATP (3-5).Nucleotide receptors responsive to both adenine and uracil nucleotidesare the P2Y₂ receptor, activated equipotently by ATP and UTP (6, 7) aswell as the Xenopus P2Y₈ (8) and turkey tp2y receptor (9) activatedequally by all triphosphate nucleotides. There are alsopyrimidinoceptors: the chicken P2Y₃ (10) and human P2Y₆ (11-13)receptors activated preferentially by UDP, and the human P2Y₄ receptor(13-15) activated preferentially by UTP. All these P2Y subtypes arecoupled to the phosphoinositide pathway. The P2Y₁₁ and tp2y receptorsare additionally coupled respectively to stimulation and inhibition ofadenylyl cyclase. Other receptors (P2Y₅ (16), P2Y₇ (17), P2Y₉ and P2Y₁₀)have been mistakenly included in the P2Y family (18-20). Recently, aP2Y₁₂ subtype has been cloned which corresponds to the platelet ADPreceptor previously called P_(2T) (21, 22). It is coupled to aninhibition of adenylyl cyclase and is specifically expressed in theplatelets and the brain. Its primary structure is not related to theother P2Y receptors but is related to that of the UDP-glucose receptor(23).

More than 300 G protein coupled receptors (GPCRs) have been cloned thusfar and it is generally assumed that well over 1000 such receptorsexist. Mechanistically, approximately 30-50% of all clinically relevantdrugs act by modulating the functions of various GPCRs (34).

Known and unknown GPCRs now constitute major targets for drug action anddevelopment.

GPR86 is a member of the rhodopsin-like receptor family, cloned in 1997(24). It shows a homology of 49% with the recently identified plateletADP receptor, P2T.

The identified ORF of 1002 bp of said receptor is preceded by a stopcodon 18 bp upstream, and the putative poly(A) signal AATAAA is present1672 bp downstream of the coding sequence. hGPR86 has the same genomiclocalization as hGPR87 on chromosome 3q24, but in contrast to hGPR87,its coding sequence is intronless. The deduced 333 amino acid residuesequence of hGPR86 shows the typical 7 transmembrane (7TM) structure ofa GPCR, with no signal peptide. It exhibits essentially the same motifsas described for GPR87 and KIAA0001, and therefore is also a member offamily 1 GPCRs. Instead of the DRY motif there is a DRF motif presentwhich is also seen in the sequences of purinergic receptors, the C5A andBonzo receptors, and the thrombin receptor precursors.

SUMMARY OF THE INVENTION

The present invention is related to the GPR86 (P2Y₁₃) receptor(identified hereafter as SEQ ID NO. 1) (or any homologous sequence) anda recombinant cell (transformed by a suitable vector) comprising thenucleotide sequence encoding the receptor, as well as the naturalligands (ADP and equivalent molecules such as 2MeSADP, ADPβS includingany of the ADP analogues presented in U.S. Pat. No. 5,700,786) to beused in screening assays for identification of agonists, inverseagonists or antagonist compounds useful for the development of new drugsand the improvement of various disease diagnostics.

A homologous sequence (which may exist in other mammal species orspecific groups of human populations), where homology indicates sequenceidentity, means a sequence which presents a high sequence identity (morethan 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) with thecomplete human nucleotide or amino acid sequence described hereafter,and is preferably characterized by the same pharmacology, especially apreference for binding to ADP>>IDP>UDP (the affinity of ADP for GPR86was approximately 1000-fold greater than that of IDP and UDP(ADP>IDP>UDP)).

Preferably, the recombinant cell according to the invention is arecombinant cell transformed by a plasmid or viral vector, preferably abaculovirus, an adenovirus, a semliki forest virus, and the cell ispreferably selected from the group consisting of bacterial cells, yeastcells, insect cells or mammal cells.

According to a preferred embodiment of the present invention, the cellis selected from the group consisting of COS-7 cells, a CHO cell, a LM(TK−) cell, a NIH-3T3 cell, HEK-293 cell, K-562 cell or a 1321N1astrocytoma cell but also other transfectable cell lines. Preferably,the vector comprises all the regulatory elements, operatively linked tothe polynucleotide sequence encoding the receptor according to theinvention so as to permit expression thereof.

Another aspect of the present invention is related to the use of aspecific active portion of the sequences. As used herein, an “activeportion” refers to a portion of a sequence that is of sufficient size toexhibit normal or near normal pharmacology (e.g., receptor activity (asdefined herein), the response to an activator or inhibitor, or ligandbinding are at least 90% of the level of activity, response, or bindingexhibited by a wild type receptor). “A portion” as it refers to asequence encoding a receptor, refers to less than 100% of the sequence(i.e., 99, 90, 80, 70, 60, 50% etc . . . ). The active portion could bea receptor which comprises a partial deletion of the complete nucleotideor amino acid sequence and which still maintains the active site(s) andprotein domain(s) necessary for the binding of and interaction with aspecific ligand, preferably ADP.

In another embodiment of any of the preceding methods, the contacting isperformed in or on synthetic liposomes (see Tajib Mirzabekov, HarryKontos, Michael Farzan, Wayne Marasco, Joseph Sodroski (2000)Paramagnetic proteoliposomes containing a pure, native, and orientedseven-transmembrane segment protein, CCR5. Nature Biotechnology 18,649-654, which is incorporated herein by reference) or virus-inducedbudding membranes containing a GPR86 polypeptide. (See Patentapplication WO0102551, Virus-like particles, their Preparation and theirUse preferably in Pharmaceutical Screening and Functional Genomics(2001) incorporated herein by references.)

As used herein, “ligand” refers to a moiety that is capable ofassociating or binding to a receptor. According to the method of theinvention, a ligand and a receptor have a binding constant that issufficiently strong to allow detection of binding by an assay methodthat is appropriate for detection of a ligand binding to a receptor(e.g. a second messenger assay to detect an increase or decrease in theproduction of a second messenger in response to ligand binding to thereceptor, a binding assay to measure protein-ligand binding or animmunoassay to measure antibody-antigen interactions). A ligandaccording to the invention includes the actual molecule that binds areceptor (e.g. ADP is the ligand for GPR86) or a ligand may be anynucleotide, antibody, antigen, enzyme, peptide, polypeptide or nucleicacid capable of binding to the receptor. A ligand is preferably anucleotide but can also include a polypeptide, a peptide or a nucleicacid sequence. According to the method of the invention, a ligand andreceptor specifically bind to each other (e.g. via covalent or hydrogenbonding or via an interaction between, for example, a protein and aligand, an antibody and an antigen or protein subunits).

As used herein, “ADP” refers to a nucleotide that is produced byhydrolysis of the terminal phosphate of ATP and has a structurecomprising adenine, ribose and two phosphate groups (FIG. 7). It iscontemplated that analogs of ADP will be considered as ADP equivalents.ADP analogs according to the invention include 2MeSADP, ADPβS. An ADPanalog according to the invention will exhibit the same basic structureas ADP, defined above and presented in FIG. 7, as well as one or moredifferent substituent groups including but not limited to any of the ADPanalogues presented in U.S. Pat. No. 5,700,786. An ADP analog accordingto the invention will exhibit binding to GPR86 that is equivalent toADP.

As used herein, “GPR activity” refers to the activity of a receptorcomprising the sequence presented in FIG. 1, or a sequence that exhibitsat least 70% identity (for example, 70%, 75%, 80%, 90%, 95% etc . . . )with the sequence presented in FIG. 1. A receptor that has “GPRactivity” will bind to ADP with an affinity that is at least 100-fold,preferably 500-fold and most preferably 1000-fold greater than that ofIDP and UDP (ADP>IDP>UDP).

Homologous sequences of a sequence according to the invention mayinclude an amino acid or nucleotide sequence encoding a similar receptorwhich exists in other animal species (rat, mouse, cat, dog, etc.) or inspecific human population groups, but which are involved in the samebiochemical pathway.

Such homologous sequences may comprise additions, deletions orsubstitutions of one or more amino acids or nucleotides, which do notsubstantially alter the functional characteristics of the receptoraccording to the invention.

Such homologous sequences can also be nucleotide sequences of more than400, 600, 800 or 1000 nucleotides able to hybridize to the completehuman sequence under stringent hybridisation conditions (such as theones described by SAMBROOK et al., Molecular Cloning, Laboratory Manuel,Cold Spring, Harbor Laboratory press, New York).

Another aspect of the present invention is related to a method for thescreening, detection and possible recovery of candidate modulators of areceptor of the invention comprising the steps of: contacting a cellexpressing GPR86 with ADP under conditions which permit binding of ADPto GPR86, in the presence of the candidate modulator, performing asecond messenger assay, and comparing the results of the secondmessenger assay obtained in the presence and absence of the candidatemodulator.

Another aspect of the present invention is related to a method for thescreening, detection and possible recovery of candidate modulators of areceptor of the invention comprising the steps of: contacting a cellmembrane expressing GPR86 with ADP under conditions which permit bindingof ADP to GPR86 performing a second messenger assay, and comparing theresults of the second messenger assay obtained in the presence andabsence of the candidate modulator.

In another embodiment, a candidate modulator or compound is selectedfrom the group consisting of a natural or synthetic peptide, apolypeptide, an antibody or antigen-binding fragment thereof, a lipid, acarbohydrate, a nucleic acid, and a small organic molecule.

In another embodiment, the step of measuring a signalling activity ofthe GPR86 polypeptide comprises detecting a change in the level of asecond messenger.

A further aspect of the present invention is related to the unknownagonist and/or antagonist compounds identified and/or recovered by themethod of the invention, as well as to a diagnostic kit comprising said(unknown) compounds or a pharmaceutical composition (including avaccine) comprising an adequate pharmaceutical carrier and a sufficientamount of said (unknown) compound.

An antagonist compound according to the invention means a molecule or agroup of molecules able to bind to the receptor according to theinvention and block the binding of natural compounds, such as ADP or anequivalent molecule, for example 2MeSADP or ADPβS, and including but notlimited to any of the ADP analogues presented in U.S. Pat. No.5,700,786.

The invention further encompasses a method of detecting the presence, ina sample, of an agent that modulates the function of GPR86, the methodcomprising: a) contacting a GPR86 polypeptide with the sample; b)detecting a signalling activity of the GPR86 polypeptide in the presenceof the sample; and c) comparing the activity measured in the presence ofthe sample to the activity measured in a reaction with GPR86 polypeptideand ADP at EC50, wherein an agent that modulates the function of GPR86is detected if the amount of the GPR86-specific activity measured in thepresence of the sample is at least 10% that of the amount induced by ADPpresent at its EC50.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of GPR86 signalling, the methodcomprising: a) contacting a tissue sample with an antibody specific fora GPR86 polypeptide; b) detecting binding of the antibody to the tissuesample; and c) comparing the binding detected in step (b) with astandard, wherein a difference in binding relative to the standard isdiagnostic of a disease or disorder characterized by dysregulation ofGPR86.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of GPR86 signalling, the methodcomprising: a) contacting a tissue sample with an antibody specific fora GPR86 ligand; b) detecting binding of the antibody to the tissuesample; and c) comparing the binding detected in step (b) with astandard, wherein a difference in binding relative to the standard isdiagnostic of a disease or disorder characterized by dysregulation ofGPR86.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of GPR86 signalling, the methodcomprising: a) contacting a tissue sample with an antibody specific fora GPR86 polypeptide and an antibody specific for a GPR86 ligand; b)detecting binding of the antibodies to the tissue sample; and c)comparing the binding detected in step (b) with a standard, wherein adifference in binding of either antibody or both, relative to thestandard, is diagnostic of a disease or disorder characterized bydysregulation of GPR86.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of GPR86 signalling, the methodcomprising: a) isolating nucleic acid from a tissue sample; b)amplifying a GPR86 polynucleotide, using the nucleic acid as a template;and c) comparing the amount of amplified GPR86 polynucleotide producedin step (b) with a standard, wherein a difference in the amount ofamplified GPR86 polynucleotide relative to the standard is diagnostic ofa disease or disorder characterized by dysregulation of GPR86.

In a preferred embodiment, the step of amplifying comprises RT/PCR. Inanother preferred embodiment, the standard is SEQ ID NO: 1. In anotherpreferred embodiment, the step of comparing the sequence comprisesminisequencing. In another preferred embodiment, the step of comparingthe amount is performed on a microarray.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of GPR86 signalling, the methodcomprising: a) isolating nucleic acid from a tissue sample; b)amplifying a polynucleotide that encodes a GPR86-specific polypeptideligand, using the nucleic acid as a template; and c) comparing theamount of amplified GPR86-specific ligand polynucleotide produced instep (b) with a standard, wherein a difference in the amount ofamplified GPR86-specific ligand polynucleotide relative to the standardis diagnostic of a disease or disorder characterized by dysregulation ofGPR86.

In a preferred embodiment, the step of amplifying comprises RT/PCR. Inanother preferred embodiment, the step of comparing the sequencecomprises minisequencing. In another preferred embodiment, the step ofcomparing the sequence is performed on a microarray.

A further aspect of the present invention is related to a transgenicnon-human mammal, comprising a homologous recombination (knock-out) ofthe polynucleotide encoding the GPR86 (P2Y₁₃) receptor according to theinvention or a transgenic non-human mammal over expressing thepolypeptide above the natural level of expression. As used herein,“above the natural level of expression” refers to a level that is atleast 2-fold, preferably 5-fold, more preferably 10-fold and mostpreferably 100-fold or more (i.e., 150-fold, 200-fold, 250-fold,500-fold, 1000-fold, 10,000-fold etc.) as compared to the level ofexpression of the endogenous receptor. A transgenic non-human mammal canbe obtained by a method well known by a person skilled in the art, forinstance, as described in document WO 98/20112 using the classicaltechnique based upon the transfection of embryonic stem cells,preferably according to the method described by Carmeliet et al.(Nature, Vol. 380, p. 435-439, 1996).

“Gene targeting” is a type of homologous recombination that occurs whena fragment of genomic DNA is introduced into a mammalian cell and thatfragment locates and recombines with endogenous homologous sequences asexemplified in U.S. Pat. No. 5,464,764, and U.S. Pat. No. 5,777,195, thecontents of which are hereby incorporated by reference herein in theirentireties. As used herein the term “transgenic animal” refers to anon-human animal in which one or more, and preferably essentially all,of the cells of the animal contain a transgene introduced by way ofhuman intervention, such as by transgenic techniques known in the art.The transgene can be introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus.

Preferably, the transgenic non-human mammal overexpressing thepolynucleotide encoding the GPR86 (P2Y₁₃) receptor according to theinvention comprises the polynucleotide incorporated in a DNA constructwith an inducible promoter allowing the overexpression of the receptorand possibly also tissue and cell-specific regulatory elements.

The diagnostic kit according to the invention includes at least GPR86receptor and, packaged separately, ADP and also may compriseadvantageously all the necessary means and media for performing adetection of specific binding (for example of ADP) to the GPR86 receptorof the invention and possibly correlating the detection of specificbinding to a method of monitoring of one or more of the symptoms of thediseases described hereafter.

Possibly, the kit comprises elements for a specific diagnostic or dosageof such bound compounds through high throughput screening techniques,well known to the person skilled in the art, especially the onedescribed in WO 00/02045. The high throughput screening diagnosticdosage and monitoring can be performed by using various solid supports,such as microtiter plates or biochips selected by the person skilled inthe art.

In the pharmaceutical composition according to the invention, theadequate pharmaceutical carrier is a carrier of solid, liquid, orgaseous form, which can be selected by the person skilled in the artaccording to the type of administration and the possible side effects ofthe compound according to the invention. The ratio between thepharmaceutical carrier and the specific compound can be selected by theperson skilled in the art according to the patient treated, theadministration and the possible side effects of the compound, as well asthe type of disease of disorder treated or submitted to a specificprevention.

-   1. The pharmaceutical composition finds advantageous applications in    the field of treatment and/or prevention of various diseases or    disorders, preferably selected from the group consisting of ostatic    hypertrophy, migraine, vomiting, psychotic and neurological    disorders, including anxiety, schizophrenia, maniac depression,    depression, delirium, dementia and severe mental retardation,    degenerative diseases, neurodegenerative diseases such as    Alzheimer's disease or Parkinson's disease, and dyskinasias, such as    Huntington's disease or Gilles de la Tourett's syndrome and other    related diseases including thrombosis and other cardiovascular    diseases, autoimmune and inflammatory diseases.-   2. Among the mentioned diseases the preferred applications are    related to therapeutic agents targeting 7TM receptors that can play    a function in preventing, improving or correcting dysfunctions or    diseases, including, but not limited to fertility, fetal    development, infections such as bacterial, fungal, protozoan and    viral infections, particularly infections caused by HIV1 and HIV2,    pain, cancer, anorexia, bulimia, asthma, Parkinson's disease, acute    heart failure, hypertension, urinary retention, osteoporosis, angina    pectoris, myocardial infarction, ulcers, asthma, allergies, benign    prostatic hypertrophy, psychotic and neurological disorders    including anxiety, depression, migraine, vomiting, stroke,    schizophrenia, manic depression, delirium, dementia, severe mental    retardation and dyskinesias, such as Huntington's disease or Gilles    de la Tourette's syndrome including thrombosis and other    cardiovascular diseases, autoimmune and inflammatory diseases.

As used herein, an “antagonist” is a ligand which competitively binds tothe receptor at the same site as an agonist, but does not activate anintracellular response initiated by an active form of a receptor, andthereby inhibits the intracellular response induced by an agonist, forexample ADP, by at least 10%, preferably 15-25%, more preferably 25-50%and most preferably, 50-100%, as compared to the intracellular responsein the presence of an agonist and in the absence of an antagonist.

As used herein, an “agonist” refers to a ligand, that activates anintracellular response when it binds to a receptor at concentrationsequal or lower to ADP concentrations which induce an intracellularresponse. An agonist according to the invention may increase theintracellular response mediated by a receptor by at least 2-fold,preferably 5-fold, more preferably 10-fold and most preferably 100-foldor more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold,10,000-fold etc . . . ), as compared to the intracellular response inthe absence of agonist. An agonist, according to the invention maydecrease internalization of a cell surface receptor such that the cellsurface expression of a receptor is increased by at least 2-fold,preferably 5-fold, more preferably 10-fold and most preferably, 100-foldor more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold,10,000-fold etc . . . ), as compared to the number of cell surfacereceptors present on the surface of a cell in the absence of an agonist.In another embodiment of the invention, an agonist stablizes a cellsurface receptor and increases the cell surface expression of a receptorby at least 2-fold, preferably 5-fold, more preferably 10-fold and mostpreferably, 100-fold or more (i.e., 200-fold, 250-fold, 500-fold,1000-fold, 10,000-fold etc . . . ), as compared to the number of cellsurface receptors present on the surface of a cell in the absence ofagonist.

As used herein, an “inverse agonist” refers to a ligand which decreasesa constitutive activity of a cell surface receptor when it binds to areceptor. An inverse agonist according to the invention may decrease theconstitutive intracellular response mediated by a receptor by at least2-fold, preferably 5-fold, more preferably 10-fold and most preferably100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold,1000-fold, 10,000-fold etc . . . ), as compared to the intracellularresponse in the absence of inverse agonist.

An “inhibitor” compound according to the invention is a moleculedirected against the receptor or against the natural ligand for thereceptor that decreases the binding of the ligand to the receptor by atleast 10%, preferably 15-25%, more preferably 25-50% and mostpreferably, 50-100%, in the presence of ADP, as compared to the bindingin the presence of ADP and in the absence of inhibitor. An “inhibitor”compound of the invention can decrease the intracellular responseinduced by an agonist, for example ADP, by at least 10%, preferably15-25%, more preferably 25-50% and most preferably, 50-100%. An“inhibitor” also refers to a nucleotide sequence encoding an inhibitorcompound of the invention.

As used herein, “natural ligand” refers to a naturally occurring ligand,found in nature, which binds to a receptor in a manner that isequivalent to ADP (i.e., with an affinity for the ligand that is greaterthan the affinity of IDP and UDP (ADP>IDP>UDP). A “natural ligand” doesnot refer to an engineered ligand that is not found in nature and thatis engineered to bind to a receptor, where it did not formerly do so ina manner different, either in degree or kind, from that which it wasengineered to do, it is no longer naturally-occurring but is“non-natural” and is derived from a naturally occurring molecule.

As used herein, a “modulator” refers to any compound that increases ordecreases the cell surface expression of a receptor of the invention,increases or decreases the binding of a ligand to a receptor of theinvention, or any compound that increases or decreases the intracellularresponse initiated by an active form of the receptor of the invention,either in the presence or absence or an agonist, and in the presence ofa ligand for the receptor, for example ADP. A modulator includes anagonist, antagonist, inhibitor or inverse agonist, as defined herein. Amodulator can be a protein, a nucleic acid, an antibody or fragmentthereof, a peptide, etc. . . . Candidate modulators can be natural orsynthetic compounds, including, for example, small molecules, compoundscontained in extracts of animal, plant, bacterial or fungal cells, aswell as conditioned medium from such cells.

As used herein, the term “small molecule” refers to a compound havingmolecular mass of less than 3000 Daltons, preferably less than 2000 or1500, still more preferably less than 1000, and most preferably lessthan 600 Daltons. A “small organic molecule” is a small molecule thatcomprises carbon.

As used herein, the term “change in binding” or “change in activity” andthe equivalent terms “difference in binding” or “difference in activity”or difference in the amount of “amplified” PCR product refer to an atleast 10% increase or decrease in binding relative to the standard, orsignalling activity or mRNA levels relative to the standard in a givenassay.

As used herein, the term “dysregulation” refers to the signallingactivity of GPR86 in a sample wherein:

a) a 10% increase or decrease in the amount of GPR86 or GPR86polypeptide ligand mRNA or polypeptide levels is measured relative tothe standard, as defined herein, of a given assay, or;

b) at least a single base pair change in the GPR86 or GPR86 polypeptideligand coding sequence is detected relative to the standard, as definedherein, of a given assay and results in an alteration of GPR signallingactivity as defined in paragraphs a), c), or d), or;

c) a 10% increase or decrease in the amount of GPR86 ligand bindingactivity is measured relative to the standard, as defined herein, of agiven assay, or;

d) a 10% increase or decrease in secondary messenger assays, as definedherein, is measured relative to the standard, as defined herein, of agiven assay.

As used herein, the term “conditions permitting the binding of ADP toGPR86” refers to conditions of, for example, temperature, saltconcentration, pH and protein concentration under which ADP binds GPR86.Exact binding conditions will vary depending upon the nature of theassay, for example, whether the assay uses viable cells or only membranefraction of cells. However, because GPR86 is a cell surface proteinfavored conditions will generally include physiological salt (90 mM) andpH (about 7.0 to 8.0). Temperatures for binding can vary from 15° C. to37° C., but will preferably be between room temperature and about 30° C.The concentration of ADP and GPR86 polypeptide in a binding reactionwill also vary, but will preferably be about 0.1 nM (e.g., in a reactionwith radiolabelled tracer ADP, where the concentration is generallybelow the K_(d)) to 1 μM (e.g., ADP as competitor).

As used herein, the term “sample” refers to the source of moleculesbeing tested for the presence of an agent or modulator compound thatmodulates binding to or signalling activity of a GPR86 polypeptide. Asample can be an environmental sample, a natural extract of animal,plant yeast or bacterial cells or tissues, a clinical sample, asynthetic sample, or a conditioned medium from recombinant cells or afermentation process. The term “tissue sample” refers to a tissue thatis tested for the presence, abundance, quality or an activity of a GPR86polypeptide, a nucleic acid encoding a GPR86 polypeptide, or an agent orcompound that modifies the ligand binding or activity of a GPR86polypeptide.

As used herein, a “tissue” is an aggregate of cells that perform aparticular function in an organism. The term “tissue” as used hereinrefers to cellular material from a particular physiological region. Thecells in a particular tissue can comprise several different cell types.A non-limiting example of this would be brain tissue that furthercomprises neurons and glial cells, as well as capillary endothelialcells and blood cells, all contained in a given tissue section orsample. In addition to solid tissues, the term “tissue” is also intendedto encompass non-solid tissues, such as blood.

As used herein, the term “membrane fraction” refers to a preparation ofcellular lipid membranes comprising a GPR86 polypeptide. As the term isused herein, a “membrane fraction” is distinct from a cellularhomogenate, in that at least a portion (i.e., at least 10%, andpreferably more) of non-membrane-associated cellular constituents hasbeen removed. The term “membrane associated” refers to those cellularconstituents that are either integrated into a lipid membrane or arephysically associated with a component that is integrated into a lipidmembrane.

As used herein, the “second messenger assay” preferably comprises themeasurement of guanine nucleotide binding or exchange, adenylatecyclase, intra-cellular cAMP, intracellular inositol phosphate,intra-cellular diacylglycerol concentration, arachinoid acidconcentration, MAP kinase(s) or tyrosine kinase(s), protein kinase Cactivity, or reporter gene expression or an aequorin-based assayaccording to methods known in the art and defined herein.

As used herein, the term “second messenger” refers to a molecule,generated or caused to vary in concentration by the activation of aG-Protein Coupled Receptor, that participates in the transduction of asignal from that GPCR. Non-limiting examples of second messengersinclude cAMP, diacylglyceorl, inositol triphosphate, arachidonic acidrelease, inositol triphosphates and intracellular calcium. The term“change in the level of a second messenger” refers to an increase ordecrease of at least 10% in the detected level of a given secondmessenger relative to the amount detected in an assay performed in theabsence of a candidate modulator.

As used herein, the term “aequorin-based assay” refers to an assay forGPCR activity that measures intracellular calcium flux induced byactivated GPCRs, wherein intracellular calcium flux is measured by theluminescence of aequorin expressed in the cell.

As used herein, the term “binding” refers to the physical association ofa ligand (e.g., ADP or an antibody) with a receptor (e.g., GPR86). Asthe term is used herein, binding is “specific” if it occurs with an EC₅₀or a K_(d) of 100 nM or less, generally in the range of 100 nM to 10 μM.For example, binding is specific if the EC₅₀ or K_(d) is 100 nM, 50 nM,10 nM, 1 nM, 950 pM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650 pM, 600pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150pM, 100 pM, 75 pM, 50 pM, 25 pM or 10 pM or less.

As used herein, the term “EC₅₀,” refers to that concentration of acompound at which a given activity, including binding of ADP or otherligand and a functional activity of a GPR86 polypeptide, is 50% of themaximum for that GPR86 activity measurable using the same assay in theabsence of compound. Stated differently, the “EC₅₀” is the concentrationof compound that gives 50% activation, when 100% activation is set atthe amount of activity that does not increase with the addition of moreagonist. It should be noted that the “EC₅₀ of ADP” will vary accordingto the identity of the ADP analogue used in the assay; for example, ADPanalogues can have EC₅₀ values higher than, lower than or the same asADP. Therefore, where an ADP analogue differs from ADP, one of the skillin the art can determine the EC₅₀ for that analogue according toconventional methods. The EC₅₀ of a given ADP is measured by performingan assay for the activity of a fixed amount of GPR86 polypeptide in thepresence of doses of ADP that increase at least until the GPR86 responseis saturated or maximal, and then plotting the measured GPR86 activityversus the concentration of ADP.

As used herein, the term “saturation” refers to the concentration of ADPor other ligand at which further increases in ligand concentration failto increase the binding of ADP ligand or GRP86-specific signallingactivity.

As used herein, the term “IC₅₀” is the concentration of an antagonist orinverse agonist that reduces the maximal activation of a GPR86 receptorby 50%.

As used herein, the term “decrease in binding” refers to a decrease ofat least 10% in the amount of binding detected in a given assay with aknown or suspected modulator of GPR86 relative to binding detected in anassay lacking that known or suspected modulator.

As used herein, the term “delivering,” when used in reference to a drugor agent, means the addition of the drug or agent to an assay mixture,or to a cell in culture. The term also refers to the administration ofthe drug or agent to an animal. Such administration can be, for example,by injection (in a suitable carrier, e.g., sterile saline or water) orby inhalation, or by an oral, transdermal, rectal, vaginal, or othercommon route of drug administration.

As used herein, the term “standard” refers to a sample taken from anindividual who is not affected by a disease or disorder characterized bydysregulation of GPR86 activity. The “standard” is used as a referencefor the comparison of GPR86 mRNA levels and quality (i.e., mutant vs.wild type), as well as for the comparison of GPR86 activities.

As used herein, the term “amplifying,” when applied to a nucleic acidsequence, refers to a process whereby one or more copies of a nucleicacid sequence is generated from a template nucleic acid. A preferredmethod of “amplifying” is PCR or RT/PCR.

As used herein, the term “G-Protein coupled receptor,” or “GPCR” refersto a membrane-associated polypeptide with 7 alpha helical transmembranedomains. Functional GPCR's associate with a ligand or agonist and alsoassociate with and activate G-proteins. GPR86 is a GPCR.

As used herein, the term “antibody” is the conventional immunoglobulinmolecule, as well as fragments thereof which are also specificallyreactive with one of the subject polypeptides. Antibodies can befragmented using conventional techniques and the fragments screened forutility in the same manner as described herein below for wholeantibodies. For example, F(ab)₂ fragments can be generated by treatingantibody with pepsin. The resulting F(ab)₂ fragment can be treated toreduce disulfide bridges to produce Fab fragments. The antibody of thepresent invention is further intended to include bispecific,single-chain, and chimeric and humanised molecules having affinity for apolypeptide conferred by at least one CDR region of the antibody. Inpreferred embodiments, the antibody further comprises a label attachedthereto and able to be detected, (e.g., the label can be a radioisotope,fluorescent compound, chemiluminescent compound, enzyme, or enzymeco-factor). The antibodies, monoclonal or polyclonal and itshypervariable portion thereof (FAB, FAB″, etc.) as well as the hybridomacell producing the antibodies are a further aspect of the presentinvention which find a specific industrial application in the field ofdiagnostics and monitoring of specific diseases, preferably the oneshereafter described.

Inhibitors according to the invention include but are not limited tolabeled monoclonal or polyclonal antibodies or hypervariable portions ofthe antibodies.

As used herein, the term “transgenic animal” refers to any animal,preferably a non-human mammal, bird, fish or an amphibian, in which oneor more of the cells of the animal contain heterologous nucleic acidintroduced by way of human intervention, such as by transgenictechniques well known in the art. The nucleic acid is introduced intothe cell, directly or indirectly by introduction into a precursor of thecell, by way of deliberate genetic manipulation, such as bymicroinjection or by infection with a recombinant virus. The termgenetic manipulation does not include classical cross-breeding, or invitro fertilization, but rather is directed to the introduction of arecombinant DNA molecule. This molecule may be integrated within achromosome, or it may be extra-chromosomally replicating DNA. In thetypical transgenic animals described herein, the transgene causes cellsto express a recombinant form of one of the subject polypeptide, e.g.either agonistic or antagonistic forms. However, transgenic animals inwhich the recombinant gene is silent are also contemplated, as forexample, the FLP or CRE recombinase dependent constructs describedbelow. Moreover, “transgenic animal” also includes those recombinantanimals in which gene disruption of one or more genes is caused by humanintervention, including both recombination and antisense techniques.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 represents nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQID NO: 2) sequence of the human GPR86 (P2Y₁₃) receptor according to theinvention.

FIG. 2 is a dendrogram representing the structural relatedness of theGPR86 (P2Y₁₃) receptor with the other P2Y subtypes.

FIG. 3 represents tissue distribution of the human GPR86 (P2Y₁₃)receptor.

FIGS. 4A to 4C represent respectively:

-   -   concentration-action curves of ADP, 2MeSADP and ADPβS on IP₃        accumulation in 1321N1-Gα16 cells expressing the GPR86 (P2Y₁₃)        human receptor;    -   agonistic effects of ADP, ATP and 2MeSATP on IP₃ accumulation in        1321N1 cells expressing the GPR86 (P2Y₁₃) human receptor        together with Gα₁₆, and;    -   the effect of pertussis toxin on IP₃ accumulation induced by ADP        on 1321N1 cells expressing the GPR86 human receptor together        with Gα₁₆.

FIGS. 5A and B represent respectively a concentration-action curve ofADP on cAMP accumulation in CHO-K1 cells expressing the GPR86 (P2Y₁₃)human receptor and the effect of pertussis toxin on cAMP accumulationinduced by ADP in CHO-K1 cells expressing the GPR86 (P2Y₁₃) humanreceptor according to the invention.

FIG. 6 shows a western blot analysis of phosphorylated Erk1 and Erk2proteins in CHO-K1 cells expressing the GPR86 (P2Y₁₃) human receptoraccording to the invention.

FIG. 7 shows the structure of ADP.

FIG. 8 shows the concentration-response curve of GPR86 activation by ATPand 2MeSATP.

FIG. 9 shows the activation of GPR86 by different diadenosinepolyphosphates.

FIG. 10 shows the concentration-reponse curve of GPR86 activation byPoly[A] and Poly[A].[G].

FIG. 11 shows the concentration-response curve of GPR 86 activation byADP in the presence of the receptor antagonists RB-2, Suramine, PPADS,MRS-2179.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the discovery that ADP is a natural ligand forthe orphan G protein coupled receptor GPR86 and methods of using thebinding of this ligand to the receptor in a drug screening method. Theknown ligand and its interaction with the receptor GPR86 also providesfor the diagnosis of conditions involving dysregulated receptoractivity. The invention also relates to a kit comprising GPR86 (P2Y₁₃)and homologous sequences, its corresponding polynucleotide and/orrecombinant cells expressing the polynucleotide, to identify agonist,antagonist and inverse agonists compounds of the receptor polypeptideand/or its corresponding polynucleotide. Such kits are useful for thediagnosis, prevention and/or a treatment of various diseases anddisorders.

The invention also relates to novel agonist, antagonist and inverseagonists compounds of the receptor polypeptide and its correspondingpolynucleotide, identified according to the method of the invention.

All references referred to below and above are incorporated by referencein their entirety.

Sequences

The invention relates to the nucleotide and amino acid sequencesencoding GPR86 (presented in FIG. 1). The invention also relates tosequences that are homologous to the nucleotide and amino acid sequencesencoding GPR86.

Calculation Of Sequence Homology

Sequence identity with respect to any of the sequences presented hereincan be determined by a simple “eyeball” comparison (i.e. a strictcomparison) of any one or more of the sequences with another sequence tosee if that other sequence has, for example, at least 70% sequenceidentity to the sequence(s).

Relative sequence identity can also be determined by commerciallyavailable computer programs that can calculate % identity between two ormore sequences using any suitable algorithm for determining identity,using for example default parameters. A typical example of such acomputer program is CLUSTAL. Other computer program methods to determineidentity and similarity between two sequences include but are notlimited to the GCG program package (Devereux et al 1984 Nucleic AcidsResearch 12: 387) and FASTA (Atschul et al 1990 J Molec Biol 403-410).

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example, when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software that can perform sequence comparisons include, but arenot limited to, the BLAST package (Ausubel et al., 1995, Short Protocolsin Molecular Biology, 3rd Edition, John Wiley & Sons), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (Ausubel et al., 1999 supra, pages 7-58 to 7-60).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied.It is preferred to use the public default values for the GCG package, orin the case of other software, the default matrix, such as BLOSUM62.

Advantageously, the BLAST algorithm is employed, with parameters set todefault values. The BLAST algorithm is described in detail athttp://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporatedherein by reference. The search parameters are defined as follows, andcan be advantageously set to the defined default parameters.

Advantageously, “substantial identity” when assessed by BLAST equates tosequences which match with an EXPECT value of at least about 7,preferably at least about 9 and most preferably 10 or more. The defaultthreshold for EXPECT in BLAST searching is usually 10.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul (Karlin and Altschul 1990,Proc. Natl. Acad. Sci. USA 87:2264-68; Karlin and Altschul, 1993, Proc.Natl. Acad. Sci. USA 90:5873-7; seehttp://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements.The BLAST programs are tailored for sequence similarity searching, forexample to identify homologues to a query sequence. For a discussion ofbasic issues in similarity searching of sequence databases, see Altschulet al (1994) Nature Genetics 6:119-129.

The five BLAST programs available at http://www.ncbi.nlm.nih.gov performthe following tasks: blastp—compares an amino acid query sequenceagainst a protein sequence database; blastn—compares a nucleotide querysequence against a nucleotide sequence database; blastx—compares thesix-frame conceptual translation products of a nucleotide query sequence(both strands) against a protein sequence database; tblastn—compares aprotein query sequence against a nucleotide sequence databasedynamically translated in all six reading frames (both strands);tblastx—compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

BLAST uses the following search parameters:

HISTOGRAM—Display a histogram of scores for each search; default is yes.(See parameter H in the BLAST Manual).

DESCRIPTIONS—Restricts the number of short descriptions of matchingsequences reported to the number specified; default limit is 100descriptions. (See parameter V in the manual page).

EXPECT—The statistical significance threshold for reporting matchesagainst database sequences; the default value is 10, such that 10matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

CUTOFF—Cutoff score for reporting high-scoring segment pairs. Thedefault value is calculated from the EXPECT value (see above). HSPs arereported for a database sequence only if the statistical significanceascribed to them is at least as high as would be ascribed to a lone HSPhaving a score equal to the CUTOFF value. Higher CUTOFF values are morestringent, leading to fewer chance matches being reported. (Seeparameter S in the BLAST Manual). Typically, significance thresholds canbe more intuitively managed using EXPECT.

ALIGNMENTS—Restricts database sequences to the number specified forwhich high-scoring segment pairs (HSPs) are reported; the default limitis 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

MATRIX—Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTNand TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).The valid alternative choices include: PAM40, PAM120, PAM250 andIDENTITY. No alternate scoring matrices are available for BLASTN;specifying the MATRIX directive in BLASTN requests returns an errorresponse.

STRAND—Restrict a TBLASTN search to just the top or bottom strand of thedatabase sequences; or restrict a BLASTN, BLASTX or TBLASTX search tojust reading frames on the top or bottom strand of the query sequence.

FILTER—Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993) Computers and Chemistry 17:149-163, or segmentsconsisting of short-periodicity internal repeats, as determined by theXNU program of Clayerie & States (1993) Computers and Chemistry17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman(see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statisticallysignificant but biologically uninteresting reports from the blast output(e.g., hits against common acidic-, basic- or proline-rich regions),leaving the more biologically interesting regions of the query sequenceavailable for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted usingthe letter “N” in nucleotide sequence (e.g., “NNNNNNNNNNNNN”) and theletter “X” in protein sequences (e.g., “XXXXXXXXX”)

Filtering is only applied to the query sequence (or its translationproducts), not to database sequences. Default filtering is DUST forBLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both,when applied to sequences in SWISS-PROT, so filtering should not beexpected to always yield an effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect.

NCBI-gi—Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

Most preferably, sequence comparisons are conducted using the simpleBLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST. Insome embodiments of the present invention, no gap penalties are usedwhen determining sequence identity.

Hybridization

The present invention also encompasses nucleotide sequences that arecapable of hybridizing to the sequences presented herein, or anyfragment or derivative thereof, or to the complement of any of theabove.

Hybridization means a “process by which a strand of nucleic acid joinswith a complementary strand through base pairing” (Coombs J (1994)Dictionary of Biotechnology, Stockton Press, New York N.Y.) as well asthe process of amplification as carried out in polymerase chain reactiontechnologies as described in Dieffenbach C W and G S Dveksler (1995, PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.).

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Nucleotide sequences of the invention capable of selectively hybridizingto the nucleotide sequences presented herein, or to their complement,will be generally at least 70%, preferably at least 75%, more preferablyat least 85 or 90% and even more preferably at least 95% or 98%homologous to the corresponding nucleotide sequences presented hereinover a region of at least 20, preferably at least 25 or 30, for instanceat least 40, 60 or 100 or more contiguous nucleotides.

The term “selectively hybridizable” means that the nucleotide sequenceused as a probe is used under conditions where a target nucleotidesequence of the invention is found to hybridize to the probe at a levelsignificantly above background. The background hybridization may occurbecause of other nucleotide sequences present, for example, in the cDNAor genomic DNA library being screened. In this event, background impliesa level of signal generated by interaction between the probe and anon-specific DNA member of the library which is less than 10 fold, andpreferably less than 100 fold as intense as the specific interactionobserved with the target DNA. The intensity of interaction may bemeasured, for example, by radiolabelling the probe, e.g. with ³²P.

Also included within the scope of the present invention are nucleotidesequences that are capable of hybridizing to the nucleotide sequencespresented herein under conditions of intermediate to maximal stringency.Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm−5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, maximum stringency hybridization can be usedto identify or detect identical nucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related nucleotide sequences.

In a preferred embodiment, the present invention covers nucleotidesequences that can hybridize to one or more of the Tramell GPCRnucleotide sequences of the present invention under stringent conditions(e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na₃ Citrate pH7.0). Where the nucleotide sequence of the invention is double-stranded,both strands of the duplex, either individually or in combination, areencompassed by the present invention. Where the nucleotide sequence issingle-stranded, it is to be understood that the complementary sequenceof that nucleotide sequence is also included within the scope of thepresent invention.

The present invention also encompasses nucleotide sequences that arecapable of hybridizing to the sequences that are complementary to thesequences presented herein, or any fragment or derivative thereof.Likewise, the present invention encompasses nucleotide sequences thatare complementary to sequences that are capable of hybridizing to thesequence of the present invention. These types of nucleotide sequencesare examples of variant nucleotide sequences. In this respect, the term“variant” encompasses sequences that are complementary to sequences thatare capable of hydridizing to the nucleotide sequences presented herein.Preferably, however, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hydridizing understringent conditions (e.g., 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015Na₃ citrate pH 7.0}) to the nucleotide sequences presented herein.

Cells

A cell that is useful according to the invention is preferably selectedfrom the group consisting of bacterial cells, yeast cells, insect cellsor mammal cells.

A cell that is useful according to the invention can be any cell intowhich a nucleic acid sequence encoding a receptor according to theinvention can be introduced such that the receptor is expressed atnatural levels or above natural levels, as defined herein. Preferably areceptor of the invention that is expressed in a cell exhibits normal ornear normal pharmacology, as defined herein. Most preferably a receptorof the invention that is expressed in a cell comprises the nucleotide oramino acid sequence presented in FIG. 1 or a nucleotide or amino acidsequence that is at least 70% identical to the amino acid sequencepresented in FIG. 1. Preferably, a receptor of the invention that isexpressed in a cell will bind ADP with an affinity that is at least100-fold, preferably 500-fold and most preferably 1000-fold greater thanthe affinity for IDP and UDP.

According to a preferred embodiment of the present invention, a cell isselected from the group consisting of COS7-cells, a CHO cell, a LM (TK−)cell, a NIH-3T3 cell, HEK-293 cell, K-562 cell or a 1321N1 astrocytomacell but also other transfectable cell lines.

Assays for the Identification of Agents that Modulate the Activity ofGPR86

Agents that modulate the activity of GPR86 can be identified in a numberof ways that take advantage of the interaction of the receptor with ADP.For example, the ability to reconstitute GPR86/ADP binding either invitro, on cultured cells or in vivo provides a target for theidentification of agents that disrupt that binding. Assays based ondisruption of binding can identify agents, such as small organicmolecules, from libraries or collections of such molecules.Alternatively, such assays can identify agents in samples or extractsfrom natural sources, e.g., plant, fungal or bacterial extracts or evenin human tissue samples (e.g., tumor tissue). In one aspect, theextracts can be made from cells expressing a library of variant nucleicacids, peptides or polypeptides. Modulators of GPR86/ADP binding canthen be screened using a binding assay or a functional assay thatmeasures downstream signalling through the receptor.

Another approach that uses the GPR86/ADP interaction more directly toidentify agents that modulate GPR86 function measures changes in GPR86downstream signalling induced by candidate agents or candidatemodulators. These functional assays can be performed in isolated cellmembrane fractions or on cells expressing the receptor on theirsurfaces.

The discovery that ADP is a ligand of the GPR86 receptor permitsscreening assays to identify agonists, antagonists and inverse agonistsof receptor activity. The screening assays will have two generalapproaches.

1) Ligand binding assays, in which cells expressing GPR86, membraneextracts from such cells, or immobilized lipid membranes comprisingGPR86 are exposed to labelled ADP and candidate compound. Followingincubation, the reaction mixture is measured for specific binding of thelabelled ADP to the GPR86 receptor. Compounds that interfere withbinding or displace labelled ADP can be agonists, antagonists or inverseagonists of GPR86 activity. Subsequent functional analysis can then beperformed on positive compounds to determine in which of thesecategories they belong.

2) Functional assays, in which a signalling activity of GPR86 ismeasured.

a) For agonist screening, cells expressing GPR86 or membranes preparedfrom them are incubated with a candidate compound, and a signallingactivity of GPR86 is measured. The activity induced by compounds thatmodulate receptor activity is compared to that induced by ADP. Anagonist or partial agonist will have a maximal biological activitycorresponding to at least 10% of the maximal activity of ADP when theagonist or partial agonist is present at 10 nM or less, and preferablywill have a potency which is at least as potent than ADP.

b) For antagonist or inverse agonist screening, cells expressing GPR86or membranes isolated from them are assayed for signalling activity inthe presence of ADP with or without a candidate compound. Antagonistswill reduce the level of ADP-stimulated receptor activity by at least10%, relative to reactions lacking the antagonist in the presence ofADP. Inverse agonists will reduce the constitutive activity of thereceptor by at least 10%, relative to reactions lacking the inverseagonist.

c) For inverse agonist screening, cells expressing constitutive GPR86activity or membranes isolated from them are used in a functional assaythat measures an activity of the receptor in the presence of a candidatecompound. Inverse agonists are those compounds that reduce theconstitutive activity of the receptor by at least 10%. Overexpression ofGPR86 may lead to constitutive activation. GPR86 can be overexpressed byplacing it under the control of a strong constitutive promoter, e.g.,the CMV early promoter. Alternatively, certain mutations of conservedGPCR amino acids or amino acid domains tend to lead to constitutiveactivity. See for example: Kjelsberg et al., 1992, J. Biol. Chem.267:1430; McWhinney et al., 2000. J. Biol. Chem. 275:2087; Ren et al.,1993, J. Biol. Chem. 268:16483; Samama et al., 1993, J. Biol. Chem268:4625; Parma et al., 1993, Nature 365:649; Parma et al., 1998, J.Pharmacol. Exp. Ther. 286:85; and Parent et al., 1996, J. Biol. Chem.271:7949.

Ligand Binding and Displacement Assays:

One can use GPR86 polypeptides expressed on a cell, or isolatedmembranes containing receptor polypeptides, along with ADP in order toscreen for compounds that inhibit the binding of ADP to GPR86. Whenidentified in an assay that measures binding or ADP displacement alone,compounds will have to be subjected to functional testing to determinewhether they act as agonists, antagonists or inverse agonists.

For displacement experiments, cells expressing a GPR86 polypeptide(generally 25,000 cells per assay or 1 to 100 μg of membrane extracts)are incubated in binding buffer with labelled ADP in the presence orabsence of increasing concentrations of a candidate modulator. Tovalidate and calibrate the assay, control competition reactions usingincreasing concentrations of unlabeled ADP can be performed. Afterincubation, cells are washed extensively, and bound, labelled ADP ismeasured as appropriate for the given label (e.g., scintillationcounting, fluorescence, etc.). A decrease of at least 10% in the amountof labelled ADP bound in the presence of candidate modulator indicatesdisplacement of binding by the candidate modulator. Candidate modulatorsare considered to bind specifically in this or other assays describedherein if they displace 50% of labelled ADP (sub-saturating ADP dose) ata concentration of 10 nM or less.

Alternatively, binding or displacement of binding can be monitored bysurface plasmon resonance (SPR). Surface plasmon resonance assays can beused as a quantitative method to measure binding between two moleculesby the change in mass near an immobilized sensor caused by the bindingor loss of binding of ADP from the aqueous phase to a GPR86 polypeptideimmobilized in a membrane on the sensor. This change in mass is measuredas resonance units versus time after injection or removal of the ADP orcandidate modulator and is measured using a Biacore Biosensor (BiacoreAB). GPR86 can be immobilized on a sensor chip (for example, researchgrade CM5 chip; Biacore AB) in a thin film lipid membrane according tomethods described by Salamon et al. (Salamon et al., 1996, Biophys J.71: 283-294; Salamon et al., 2001, Biophys. J. 80: 1557-1567; Salamon etal., 1999, Trends Biochem. Sci. 24: 213-219, each of which isincorporated herein by reference.). Sarrio et al. demonstrated that SPRcan be used to detect ligand binding to the GPCR A(1) adenosine receptorimmobilized in a lipid layer on the chip (Sarrio et al., 2000, Mol.Cell. Biol. 20: 5164-5174, incorporated herein by reference). Conditionsfor ADP binding to GPR86 in an SPR assay can be fine-tuned by one ofskill in the art using the conditions reported by Sarrio et al. as astarting point.

SPR can assay for modulators of binding in at least two ways. First, ADPcan be pre-bound to immobilized GPR86 polypeptide, followed by injectionof candidate modulator at a concentration ranging from 0.1 nM to 1 μM.Displacement of the bound ADP can be quantitated, permitting detectionof modulator binding. Alternatively, the membrane-bound GPR86polypeptide can be pre-incubated with candidate modulator and challengedwith ADP. A difference in ADP binding to the GPR86 exposed to modulatorrelative to that on a chip not pre-exposed to modulator will demonstratebinding or displacement of ADP in the presence of modulator. In eitherassay, a decrease of 10% or more in the amount of ADP bound is in thepresence of candidate modulator, relative to the amount of a ADP boundin the absence of candidate modulator indicates that the candidatemodulator inhibits the interaction of GPR86 and ADP.

Another method of detecting inhibition of binding of ADP to GPR86 usesfluorescence resonance energy transfer (FRET). FRET is a quantummechanical phenomenon that occurs between a fluorescence donor (D) and afluorescence acceptor (A) in close proximity to each other (usually <100A of separation) if the emission spectrum of D overlaps with theexcitation spectrum of A. The molecules to be tested, e.g. ADP and aGPR86 polypeptide, are labelled with a complementary pair of donor andacceptor fluorophores. While bound closely together by the GPR86:ADPinteraction, the fluorescence emitted upon excitation of the donorfluorophore will have a different wavelength than that emitted inresponse to that excitation wavelength when the ADP and GPR86polypeptide are not bound, providing for quantitation of bound versusunbound molecules by measurement of emission intensity at eachwavelength. Donor fluorophores with which to label the GPR86 polypeptideare well known in the art. Of particular interest are variants of the A.victoria GFP known as Cyan FP(CFP, Donor (D)) and Yellow FP (YFP,Acceptor(A)). As an example, the YFP variant can be made as a fusionprotein with GPR86. Vectors for the expression of GFP variants asfusions (Clontech) as well as flurophore-labeled ADP compounds(Molecular Probes) are known in the art. The addition of a candidatemodulator to the mixture of labelled ADP and YFP-GPR86 protein willresult in an inhibition of energy transfer evidenced by, for example, adecrease in YFP fluorescence relative to a sample without the candidatemodulator. In an assay using FRET for the detection of GPR86:ADPinteraction, a 10% or greater decrease in the intensity of fluorescentemission at the acceptor wavelength in samples containing a candidatemodulator, relative to samples without the candidate modulator,indicates that the candidate modulator inhibits the GPR86:ADPinteraction.

A variation on FRET uses fluorescence quenching to monitor molecularinteractions. One molecule in the interacting pair can be labelled witha fluorophore, and the other with a molecule that quenches thefluorescence of the fluorophore when brought into close apposition withit. A change in fluorescence upon excitation is indicative of a changein the association of the molecules tagged with the fluorophore:quencherpair. Generally, an increase in fluorescence of the labelled GPR86polypeptide is indicative that the ADP molecule bearing the quencher hasbeen displaced. For quenching assays, a 10% or greater increase in theintensity of fluorescent emission in samples containing a candidatemodulator, relative to samples without the candidate modulator,indicates that the candidate modulator inhibits GPR86:ADP interaction.

In addition to the surface plasmon resonance and FRET methods,fluorescence polarization measurement is useful to quantitate binding.The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Complexes,such as those formed by GPR86 associating with a fluorescently labelledADP, have higher polarization values than uncomplexed, labelled ADP. Theinclusion of a candidate inhibitor of the GPR86:ADP interaction resultsin a decrease in fluorescence polarization, relative to a mixturewithout the candidate inhibitor, if the candidate inhibitor disrupts orinhibits the interaction of GPR86 with ADP. Fluorescence polarization iswell suited for the identification of small molecules that disrupt theformation of receptor:ligand complexes. A decrease of 10% or more influorescence polarization in samples containing a candidate modulator,relative to fluorescence polarization in a sample lacking the candidatemodulator, indicates that the candidate modulator inhibits GPR86:ADPinteraction.

Another alternative for monitoring GPR86:ADP interactions uses abiosensor assay. ICS biosensors have been described in the art(Australian Membrane Biotechnology Research Institute;http//www.ambri.com.au/; Cornell B, Braach-Maksvytis V, King L, Osman P,Raguse B, Wieczorek L, and Pace R. “A biosensor that uses ion-channelswitches” Nature 1997, 387, 580). In this technology, the association ofGPR86 and its ligand, is coupled to the closing ofgramacidin-facilitated ion channels in suspended membrane bilayers andthus to a measurable change in the admittance (similar to impedence) ofthe biosensor. This approach is linear over six orders of magnitude ofadmittance change and is ideally suited for large scale, high throughputscreening of small molecule combinatorial libraries. A 10% or greaterchange (increase or decrease) in admittance in a sample containing acandidate modulator, relative to the admittance of a sample lacking thecandidate modulator, indicates that the candidate modulator inhibits theinteraction of GPR86 and ADP. It is important to note that in assaystesting the interaction of GPR86 with ADP, it is possible that amodulator of the interaction need not necessarily interact directly withthe domain(s) of the proteins that physically interact with ADP. It isalso possible that a modulator will interact at a location removed fromthe site of interaction and cause, for example, a conformational changein the GPR86 polypeptide. Modulators (inhibitors or agonists) that actin this manner are nonetheless of interest as agents to modulate theactivity of GPR86.

-   3. It should be understood that any of the binding assays described    herein can be performed with a non-ADP ligand (for example, agonist,    antagonist, etc.) of GPR86, e.g., a small molecule identified as    described herein or ADP analogues including but not limited to any    of the ADP analogues presented in U.S. Pat. No. 5,700,786, a natural    or synthetic peptide, a polypeptide, an antibody or antigen-binding    fragment thereof, a lipid, a carbohydrate, and a small organic    molecule.

Any of the binding assays described can be used to determine thepresence of an agent in a sample, e.g., a tissue sample, that binds tothe GPR86 receptor molecule, or that affects the binding of ADP to thereceptor. To do so, GPR86 polypeptide is reacted with ADP or anotherligand in the presence or absence of the sample, and ADP or ligandbinding is measured as appropriate for the binding assay being used. Adecrease of 10% or more in the binding of ADP or other ligand indicatesthat the sample contains an agent that modulates ADP or ligand bindingto the receptor polypeptide.

Functional Assays of Receptor Activity

i. GTPase/GTP Binding Assays:

For GPCRs such as GPR86, a measure of receptor activity is the bindingof GTP by cell membranes containing receptors. In the method describedby Traynor and Nahorski, 1995, Mol. Pharmacol. 47: 848-854, incorporatedherein by reference, one essentially measures G-protein coupling tomembranes by detecting the binding of labelled GTP. For GTP bindingassays, membranes isolated from cells expressing the receptor areincubated in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, and10 mM MgCl2, 80 pM ³⁵S-GTPγS and 3 μM GDP. The assay mixture isincubated for 60 minutes at 30° C., after which unbound labelled GTP isremoved by filtration onto GF/B filters. Bound, labelled GTP is measuredby liquid scintillation counting. In order to assay for modulation ofADP-induced GPR86 activity, membranes prepared from cells expressing aGPR86 polypeptide are mixed with ADP, and the GTP binding assay isperformed in the presence and absence of a candidate modulator of GPR86activity. A decrease of 10% or more in labelled GTP binding as measuredby scintillation counting in an assay of this kind containing acandidate modulator, relative to an assay without the modulator,indicates that the candidate modulator inhibits GPR86 activity. Asimilar GTP-binding assay can be performed without ADP to identifycompounds that act as agonists. In this case, ADP-stimulated GTP bindingis used as a standard. A compound is considered an agonist if it inducesat least 50% of the level of GTP binding induced by ADP when thecompound is present at 1 μM or less, and preferably will induce a levelthe same as or higher than that induced by ADP.

GTPase activity is measured by incubating the membranes containing aGPR86 polypeptide with γ³²P-GTP. Active GTPase will release the label asinorganic phosphate, which is detected by separation of free inorganicphosphate in a 5% suspension of activated charcoal in 20 mM H₃PO₄,followed by scintillation counting. Controls include assays usingmembranes isolated from cells not expressing GPR86 (mock-transfected),in order to exclude possible non-specific effects of the candidatecompound.

In order to assay for the effect of a candidate modulator onGPR86-regulated GTPase activity, membrane samples are incubated withADP, with and without the modulator, followed by the GTPase assay. Achange (increase or decrease) of 10% or more in the level of GTP bindingor GTPase activity relative to samples without modulator is indicativeof GPR86 modulation by a candidate modulator.

ii. Downstream Pathway Activation Assays:

a. Calcium Flux—The Aeguorin-Based Assay:

The aequorin assay takes advantage of the responsiveness ofmitochondrial apoaequorin to intracellular calcium release induced bythe activation of GPCRs (Stables et al., 1997, Anal. Biochem.252:115-126; Detheux et al., 2000, J. Exp. Med., 192 1501-1508; both ofwhich are incorporated herein by reference). Briefly, GPR86-expressingclones are transfected to coexpress mitochondrial apoaequorin and Gα16.Cells are incubated with 5 μM Coelenterazine H (Molecular Probes) for 4hours at room temperature, washed in DMEM-F12 culture medium andresuspended at a concentration of 0.5×10⁶ cells/ml. Cells are then mixedwith test agonist molecules and light emission by the aequorin isrecorded with a luminometer for 30 sec. Results are expressed asRelative Light Units (RLU). Controls include assays using membranesisolated from cells not expressing GPR86 (mock transfected), in order toexclude possible non-specific effects of the candidate compound.

Aequorin activity or intracellular calcium levels are “changed” if lightintensity increases or decreases by 10% or more in a sample of cells,expressing a GPR86 polypeptide and treated with a candidate modulator,relative to a sample of cells expressing the GPR86 polypeptide but nottreated with the candidate modulator or relative to a sample of cellsnot expressing the GPR86 polypeptide (mock-transfected cells) buttreated with the candidate modulator.

When performed in the absence of ADP, the assay can be used to identifyan agonist of GPR86 activity. When the assay is performed in thepresence of ADP, it can be used to assay for an antagonist.

b. Adenylate Cyclase Assay:

Assays for adenylate cyclase activity are described by Kenimer &Nirenberg, 1981, Mol. Pharmacol. 20: 585-591, incorporated herein byreference. That assay is a modification of the assay taught by Solomonet al., 1974, Anal. Biochem. 58: 541-548, also incorporated herein byreference. Briefly, 100 μl reactions contain 50 mM Tris-Hcl (pH 7.5), 5mM MgCl₂, 20 mM creatine phosphate (disodium salt), 10 units (71 μg ofprotein) of creatine phosphokinase, 1 mM α-³²P-ATP (tetrasodium salt, 2μCi), 0.5 mM cyclic AMP, G-³H-labeled cyclic AMP (approximately 10,000cpm), 0.5 mM Ro20-1724, 0.25% ethanol, and 50-200 μg of proteinhomogenate to be tested (i.e., homogenate from cells expressing or notexpressing a GPR86 polypeptide, treated or not treated with ADP with orwithout a candidate modulator). Reaction mixtures are generallyincubated at 37° C. for 6 minutes. Following incubation, reactionmixtures are deproteinized by the addition of 0.9 ml of cold 6%trichloroacetic acid. Tubes are centrifuged at 1800×g for 20 minutes andeach supernatant solution is added to a Dowex AG50W-X4 column. The cAMPfraction from the column is eluted with 4 ml of 0.1 mM imidazole-HCl (pH7.5) into a counting vial. Assays should be performed in triplicate.Control reactions should also be performed using protein homogenate fromcells that do not express a GPR86 polypeptide.

According to the invention, adenylate cyclase activity is “changed” ifit increases or decreases by 10% or more in a sample taken from cellstreated with a candidate modulator of GPR86 activity, relative to asimilar sample of cells not treated with the candidate modulator orrelative to a sample of cells not expressing the GPR86 polypeptide(mock-transfected cells) but treated with the candidate modulator.

c. cAMP Assay:

Intracellular or extracellular cAMP is measured using a cAMPradioimmunoassay (RIA) or cAMP binding protein according to methodswidely known in the art. For example, Horton & Baxendale, 1995, MethodsMol. Biol. 41: 91-105, which is incorporated herein by reference,describes an RIA for cAMP.

A number of kits for the measurement of cAMP are commercially available,such as the High Efficiency Fluorescence Polarization-based homogeneousassay marketed by LJL Biosystems and NEN Life Science Products. Controlreactions should be performed using extracts of mock-transfected cellsto exclude possible non-specific effects of some candidate modulators.

The level of cAMP is “changed” if the level of cAMP detected in cells,expressing a GPR86 polypeptide and treated with a candidate modulator ofGPR86 activity (or in extracts of such cells), using the RIA-based assayof Horton & Baxendale, 1995, supra, increases or decreases by at least10% relative to the cAMP level in similar cells not treated with thecandidate modulator.

d. Phospholipid Breakdown, DAG Production and Inositol TriphosphateLevels:

Receptors that activate the breakdown of phospholipids can be monitoredfor changes due to the activity of known or suspected modulators ofGPR86 by monitoring phospholipid breakdown, and the resulting productionof second messengers DAG and/or inositol triphosphate (IP₃). Methods ofdetecting each of these are described in Phospholipid SignallingProtocols, edited by Ian M. Bird. Totowa, N.J., Humana Press, 1998,which is incorporated herein by reference. See also Rudolph et al.,1999, J. Biol. Chem. 274: 11824-11831, incorporated herein by reference,which also describes an assay for phosphatidylinositol breakdown. Assaysshould be performed using cells or extracts of cells expressing GPR86,treated or not treated with ADP with or without a candidate modulator.Control reactions should be performed using mock-transfected cells, orextracts from them in order to exclude possible non-specific effects ofsome candidate modulators.

According to the invention, phosphatidylinositol breakdown, anddiacylglycerol and/or inositol triphosphate levels are “changed” if theyincrease or decrease by at least 10% in a sample from cells expressing aGPR86 polypeptide and treated with a candidate modulator, relative tothe level observed in a sample from cells expressing a GPR86 polypeptidethat is not treated with the candidate modulator.

e. PKC Activation Assays:

Growth factor receptor tyrosine kinases can signal via a pathwayinvolving activation of Protein Kinase C (PKC), which is a family ofphospholipid- and calcium-activated protein kinases. PKC activationultimately results in the transcription of an array of proto-oncogenetranscription factor-encoding genes, including c-fos, c-myc and c-jun,proteases, protease inhibitors, including collagenase type I andplasminogen activator inhibitor, and adhesion molecules, includingintracellular adhesion molecule I (ICAM I). Assays designed to detectincreases in gene products induced by PKC can be used to monitor PKCactivation and thereby receptor activity. In addition, the activity ofreceptors that signal via PKC can be monitored through the use ofreporter gene constructs driven by the control sequences of genesactivated by PKC activation. This type of reporter gene-based assay isdiscussed in more detail below.

For a more direct measure of PKC activity, the method of Kikkawa et al.,1982, J. Biol. Chem. 257: 13341, incorporated herein by reference, canbe used. This assay measures phosphorylation of a PKC substrate peptide,which is subsequently separated by binding to phosphocellulose paper.This PKC assay system can be used to measure activity of purifiedkinase, or the activity in crude cellular extracts. Protein kinase Csample can be diluted in 20 mM HEPES/2 mM DTT immediately prior toassay.

The substrate for the assay is the peptide Ac-FKKSFKL-NH2 (SEQ ID NO:10), derived from the myristoylated alanine-rich protein kinase Csubstrate protein (MARCKS). The K_(m) of the enzyme for this peptide isapproximately 50 μM. Other basic, protein kinase C-selective peptidesknown in the art can also be used, at a concentration of at least 2-3times their K_(m). Cofactors required for the assay include calcium,magnesium, ATP, phosphatidylserine and diacylglycerol. Depending uponthe intent of the user, the assay can be performed to determine theamount of PKC present (activating conditions) or the amount of activePKC present (non-activating conditions). For most purposes according tothe invention, non-activating conditions will be used, such that thePKC, that is active in the sample when it is isolated, is measured,rather than measuring the PKC that can be activated. For non-activatingconditions, calcium is omitted from the assay in favor of EGTA.

The assay is performed in a mixture containing 20 mM HEPES, pH 7.4, 1-2mM DTT, 5 mM MgCl₂, 100 μM ATP, ˜1 μCi γ-³²P-ATP, 100 μg/ml peptidesubstrate (˜100 μM), 140 μM/3.8 μM phosphatidylserine/diacylglycerolmembranes, and 100 μM calcium (or 500 μM EGTA). 48 μl of sample, dilutedin 20 mM HEPES, pH 7.4, 2 mM DTT is used in a final reaction volume of80 μl. Reactions are performed at 30° C. for 5-10 minutes, followed byaddition of 25 μl of 100 mM ATP, 100 mM EDTA, pH 8.0, which stops thereactions.

After the reaction is stopped, a portion (85 μl) of each reaction isspotted onto a Whatman P81 cellulose phosphate filter, followed bywashes: four times 500 ml in 0.4% phosphoric acid, (5-10 min per wash);and a final wash in 500 ml 95% EtOH, for 2-5 min. Bound radioactivity ismeasured by scintillation counting. Specific activity (cpm/nmol) of thelabelled ATP is determined by spotting a sample of the reaction onto P81paper and counting without washing. Units of PKC activity, defined asnmol phosphate transferred per min, are calculated as follows:

The activity, in UNITS (nmol/min) is:$= {\frac{\left( {{cpm}\quad{on}\quad{paper}} \right) \times \left( {105\quad{\mu l}\quad{{total}/85}\quad{\mu l}\quad{spotted}} \right)}{\left( {{{assay}\quad{time}},\min} \right)\left( {{specific}\quad{activity}\quad{of}\quad{ATP}\quad{cpm}\text{/}n\quad{mol}} \right)}.}$

An alternative assay can be performed using a Protein Kinase C Assay Kitsold by PanVera (Cat. # P2747).

Assays are performed on extracts from cells expressing a GPR86polypeptide, treated or not treated with ADP with or without a candidatemodulator. Control reactions should be performed using mock-transfectedcells, or extracts from them in order to exclude possible non-specificeffects of some candidate modulators.

According to the invention, PKC activity is “changed” by a candidatemodulator when the units of PKC measured by either assay described aboveincrease or decrease by at least 10%, in extracts from cells expressingGPR86 and treated with a candidate modulator, relative to a reactionperformed on a similar sample from cells not treated with a candidatemodulator.

f. Kinase Assays:

MAP kinase activity can be assayed using any of several kits availablecommercially, for example, the p38 MAP Kinase assay kit sold by NewEngland Biolabs (Cat # 9820) or the FlashPlate™ MAP Kinase assays soldby Perkin-Elmer Life Sciences.

MAP Kinase activity is “changed” if the level of activity is increasedor decreased by 10% or more in a sample from cells, expressing a GPR86polypeptide, treated with a candidate modulator relative to MAP kinaseactivity in a sample from similar cells not treated with the candidatemodulator.

Direct assays for tyrosine kinase activity using known synthetic ornatural tyrosine kinase substrates and labelled phosphate are wellknown, as are similar assays for other types of kinases (e.g., Ser/Thrkinases). Kinase assays can be performed with both purified kinases andcrude extracts prepared from cells expressing a GPR86 polypeptide,treated with or without ADP, with or without a candidate modulator.Control reactions should be performed using mock-transfected cells, orextracts from them in order to exclude possible non-specific effects ofsome candidate modulators. Substrates can be either full-length proteinor synthetic peptides representing the substrate. Pinna & Ruzzene (1996,Biochem. Biophys. Acta 1314: 191-225, incorporated herein by reference)list a number of phosphorylation substrate sites useful for detectingkinase activities. A number of kinase substrate peptides arecommercially available. One that is particularly useful is the“Src-related peptide,” RRLIEDAEYAARG (SEQ ID NO: 11) (available fromSigma # A7433), which is a substrate for many receptor and nonreceptortyrosine kinases. Because the assay described below requires binding ofpeptide substrates to filters, the peptide substrates should have a netpositive charge to facilitate binding. Generally, peptide substratesshould have at least 2 basic residues and a free amino terminus.Reactions generally use a peptide concentration of 0.7-1.5 mM.

Assays are generally carried out in a 25 μl volume comprising 5 μl of 5×kinase buffer (5 mg/mL BSA, 150 mM Tris-Cl (pH 7.5), 100 mM MgCl₂;depending upon the exact kinase assayed for, MnCl₂ can be used in placeof or in addition to the MgCl₂), 5 μl of 1.0 mM ATP (0.2 mM finalconcentration), γ-32P-ATP (100-500 cpm/pmol), 3 μl of 10 mM peptidesubstrate (1.2 mM final concentration), cell extract containing kinaseto be tested (cell extracts used for kinase assays should contain aphosphatase inhibitor (e.g. 0.1-1 mM sodium orthovanadate)), and H₂O to25 μl. Reactions are performed at 30° C., and are initiated by theaddition of the cell extract.

Kinase reactions are performed for 30 seconds to about 30 minutes,followed by the addition of 45 μl of ice-cold 10% trichloroacetic acid(TCA). Samples are spun for 2 minutes in a microcentrifuge, and 35 μl ofthe supernatant is spotted onto Whatman P81 cellulose phosphate filtercircles. The filters are washed three times with 500 ml cold 0.5%phosphoric acid, followed by one wash with 200 ml of acetone at roomtemperature for 5 minutes. Filters are dried and incorporated ³²P ismeasured by scintillation counting. The specific activity of ATP in thekinase reaction (e.g., in cpm/pmol) is determined by spotting a smallsample (2-5 μl) of the reaction onto a P81 filter circle and countingdirectly, without washing. Counts per minute obtained in the kinasereaction (minus blank) are then divided by the specific activity todetermine the moles of phosphate transferred in the reaction.

Tyrosine kinase activity is “changed” if the level of kinase activity isincreased or decreased by 10% or more in a sample from cells, expressinga GPR86 polypeptide, treated with a candidate modulator relative tokinase activity in a sample from similar cells not treated with thecandidate modulator.

g. Transcriptional Reporters for Downstream Pathway Activation:

The intracellular signal initiated by binding of an agonist to areceptor, e.g., GPR86, sets in motion a cascade of intracellular events,the ultimate consequence of which is a rapid and detectable change inthe transcription or translation of one or more genes. The activity ofthe receptor can therefore be monitored by detecting the expression of areporter gene driven by control sequences responsive to GPR86activation.

As used herein “promoter” refers to the transcriptional control elementsnecessary for receptor-mediated regulation of gene expression, includingnot only the basal promoter, but also any enhancers ortranscription-factor binding sites necessary for receptor-regulatedexpression. By selecting promoters that are responsive to theintracellular signals resulting from agonist binding, and operativelylinking the selected promoters to reporter genes whose transcription,translation or ultimate activity is readily detectable and measurable,the transcription based reporter assay provides a rapid indication ofwhether a given receptor is activated.

Reporter genes such as luciferase, CAT, GFP, β-lactamase orβ-galactosidase are well known in the art, as are assays for thedetection of their products.

Genes particularly well suited for monitoring receptor activity are the“immediate early” genes, which are rapidly induced, generally withinminutes of contact between the receptor and the effector protein orligand. The induction of immediate early gene transcription does notrequire the synthesis of new regulatory proteins. In addition to rapidresponsiveness to ligand binding, characteristics of preferred genesuseful for making reporter constructs include: low or undetectableexpression in quiescent cells; induction that is transient andindependent of new protein synthesis; subsequent shut-off oftranscription requires new protein synthesis; and mRNAs transcribed fromthese genes have a short half-life. It is preferred, but not necessarythat a transcriptional control element have all of these properties forit to be useful.

An example of a gene that is responsive to a number of different stimuliis the c-fos proto-oncogene. The c-fos gene is activated in aprotein-synthesis-independent manner by growth factors, hormones,differentiation-specific agents, stress, and other known inducers ofcell surface proteins. The induction of c-fos expression is extremelyrapid, often occurring within minutes of receptor stimulation. Thischaracteristic makes the c-fos regulatory regions particularlyattractive for use as a reporter of receptor activation.

The c-fos regulatory elements include (see, Verma et al., 1987, Cell 51:513-514): a TATA box that is required for transcription initiation; twoupstream elements for basal transcription, and an enhancer, whichincludes an element with dyad symmetry and which is required forinduction by TPA, serum, EGF, and PMA.

The 20 bp c-fos transcriptional enhancer element located between −317and −298 bp upstream from the c-fos mRNA cap site, is essential forserum induction in serum starved NIH 3T3 cells. One of the two upstreamelements is located at −63 to −57 and it resembles the consensussequence for cAMP regulation.

The transcription factor CREB (cyclic AMP responsive element bindingprotein) is, as the name implies, responsive to levels of intracellularcAMP. Therefore, the activation of a receptor that signals viamodulation of cAMP levels can be monitored by detecting either thebinding of the transcription factor, or the expression of a reportergene linked to a CREB-binding element (termed the CRE, or cAMP responseelement). The DNA sequence of the CRE is TGACGTCA. Reporter constructsresponsive to CREB binding activity are described in U.S. Pat. No.5,919,649.

Other promoters and transcriptional control elements, in addition to thec-fos elements and CREB-responsive constructs, include the vasoactiveintestinal peptide (VIP) gene promoter (cAMP responsive; Fink et al.,1988, Proc. Natl. Acad. Sci. 85:6662-6666); the somatostatin genepromoter (cAMP responsive; Montminy et al., 1986, Proc. Natl. Acad. Sci.8.3:6682-6686); the proenkephalin promoter (responsive to cAMP,nicotinic agonists, and phorbol esters; Comb et al., 1986, Nature323:353-356); the phosphoenolpyruvate carboxy-kinase (PEPCK) genepromoter (cAMP responsive; Short et al., 1986, J. Biol. Chem.261:9721-9726).

Additional examples of transcriptional control elements that areresponsive to changes in GPCR activity include, but are not limited tothose responsive to the AP-1 transcription factor and those responsiveto NF-κB activity. The consensus AP-1 binding site is the palindromeTGA(C/G)TCA (Lee et al., 1987, Nature 325: 368-372; Lee et al., 1987,Cell 49: 741-752). The AP-1 site is also responsible for mediatinginduction by tumor promoters such as the phorbol ester12-O-tetradecanoylphorbol-β-acetate (TPA), and are therefore sometimesalso referred to as a TRE, for TPA-response element. AP-1 activatesnumerous genes that are involved in the early response of cells togrowth stimuli. Examples of AP-1-responsive genes include, but are notlimited to the genes for Fos and Jun (which proteins themselves make upAP-1 activity), Fos-related antigens (Fra) 1 and 2, IκBα, ornithinedecarboxylase, and annexins I and II.

The NF-κB binding element has the consensus sequence GGGGACTTTCC (SEQ IDNO: 3). A large number of genes have been identified as NF-κBresponsive, and their control elements can be linked to a reporter geneto monitor GPCR activity. A small sample of the genes responsive toNF-κB includes those encoding IL-1β (Hiscott et al., 1993, Mol. Cell.Biol. 13: 6231-6240), TNF-α (Shakhov et al., 1990, J. Exp. Med. 171:35-47), CCR5 (Liu et al., 1998, AIDS Res. Hum. Retroviruses 14:1509-1519), P-selection (Pan & McEver, 1995, J. Biol. Chem. 270:23077-23083), Fas ligand (Matsui et al., 1998, J. Immunol. 161:3469-3473), GM-CSF (Schreck & Baeuerle, 1990, Mol. Cell. Biol. 10:1281-1286) and IκBα (Haskill et al., 1991, Cell 65: 1281-1289). Each ofthese references is incorporated herein by reference. Vectors encodingNF-κB-responsive reporters are also known in the art or can be readilymade by one of skill in the art using, for example, synthetic NF-κBelements and a minimal promoter, or using the NF-κB-responsive sequencesof a gene known to be subject to NF-κB regulation. Further, NF-κBresponsive reporter constructs are commercially available from, forexample, CLONTECH.

A given promoter construct should be tested by exposing GPR86-expressingcells, transfected with the construct, to ADP. An increase of at leasttwo-fold in the expression of reporter in response to ADP indicates thatthe reporter is an indicator of GPR86 activity.

In order to assay GPR86 activity with an ADP responsive transcriptionalreporter construct, cells that stably express a GPR86 polypeptide arestably transfected with the reporter construct. To screen for agonists,the cells are left untreated, exposed to candidate modulators, orexposed to ADP, and expression of the reporter is measured. TheADP-treated cultures serve as a standard for the level of transcriptioninduced by a known agonist. An increase of at least 50% in reporterexpression in the presence of a candidate modulator indicates that thecandidate is a modulator of GPR86 activity. An agonist will induce atleast as much, and preferably the same amount or more, reporterexpression than ADP alone. This approach can also be used to screen forinverse agonists where cells express a GPR86 polypeptide at levels suchthat there is an elevated basal activity of the reporter in the absenceof ADP or another agonist. A decrease in reporter activity of 10% ormore in the presence of a candidate modulator, relative to its absence,indicates that the compound is an inverse agonist.

To screen for antagonists, the cells expressing GPR86 and carrying thereporter construct are exposed to ADP (or another agonist) in thepresence and absence of candidate modulator. A decrease of 10% or morein reporter expression in the presence of candidate modulator, relativeto the absence of the candidate modulator, indicates that the candidateis a modulator of GPR86 activity.

Controls for transcription assays include cells not expressing GPR86 butcarrying the reporter construct, as well as cells with a promoterlessreporter construct. Compounds that are identified as modulators ofGPR86-regulated transcription should also be analyzed to determinewhether they affect transcription driven by other regulatory sequencesand by other receptors, in order to determine the specificity andspectrum of their activity.

The transcriptional reporter assay, and most cell-based assays, are wellsuited for screening expression libraries for proteins for those thatmodulate GPR86 activity. The libraries can be, for example, cDNAlibraries from natural sources, e.g., plants, animals, bacteria, etc.,or they can be libraries expressing randomly or systematically mutatedvariants of one or more polypeptides. Genomic libraries in viral vectorscan also be used to express the mRNA content of one cell or tissue, inthe different libraries used for screening of GPR86.

h) Inositol Phosphates (IP) Measurement:

Cells of the invention, for example, 1321N1 cells, are labelled for 24hours with 10 μCi/ml [³H] inositol in inositol free DMEM containing 5%FCS, antibiotics, amphotericin, sodium pyruvate and 400 μg/ml G418.Cells are incubated for 2 h in Krebs-Ringer Hepes (KRH) buffer of thefollowing composition (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO₄, 1.45 mMCaCl₂, 1.25 mM KH₂PO₄, 25 mM Hepes (pH: 7.4) and 8 mM glucose). Thecells are then challenged with various nucleotides for 30 s. Theincubation is stopped by the addition of an ice cold 3% perchloric acidsolution. IP are extracted and separated on Dowex columns as previouslydescribed (25). 2MeSATP and ATP solutions (1 mM) are treated at roomtemperature with 20 units/ml CPK and 10 Mm cp for 90 min to circumventproblems arising from the contamination and degradation of triphosphatenucleotide solutions.

GPR86 Assay

The invention provides for an assay for detecting the activity of areceptor of the invention in a sample. For example, GPR86 activity canbe measured in a sample comprising a cell or a cell membrane thatexpresses GPR86. The assay is performed by incubating the sample in thepresence or absence of ADP and carrying out a second messenger assay, asdescribed above. The results of the second messenger assay performed inthe presence or absence of ADP are compared to determine if the GPR86receptor is active. An increase of 10% or more in the detected level ofa given second messenger, as defined herein, in the presence of ADPrelative to the amount detected in an assay performed in the absence ofADP is indicative of GPR86 activity.

Any of the assays of receptor activity, including but not limited to theGTP-binding, GTPase, adenylate cyclase, cAMP, phospholipid-breakdown,diacylglycerol, inositol triphosphate, arachidonic acid release (seebelow), PKC, kinase and transcriptional reporter assays, can be used todetermine the presence of an agent in a sample, e.g., a tissue sample,that affects the activity of the GPR86 receptor molecule. To do so,GPR86 polypeptide is assayed for activity in the presence and absence ofthe sample or an extract of the sample. An increase in GPR86 activity inthe presence of the sample or extract relative to the absence of thesample indicates that the sample contains an agonist of the receptoractivity. A decrease in receptor activity in the presence of ADP oranother agonist and the sample, relative to receptor activity in thepresence of ADP alone, indicates that the sample contains an antagonistof GPR86 activity. If desired, samples can then be fractionated andfurther tested to isolate or purify the agonist or antagonist. Theamount of increase or decrease in measured activity necessary for asample to be said to contain a modulator depends upon the type of assayused. Generally, a 10% or greater change (increase or decrease) relativeto an assay performed in the absence of a sample indicates the presenceof a modulator in the sample. One exception is the transcriptionalreporter assay, in which at least a two-fold increase or 10% decrease insignal is necessary for a sample to be said to contain a modulator. Itis preferred that an agonist stimulates at least 50%, and preferably 75%or 100% or more, e.g., 2-fold, 5-fold, 10-fold or greater receptoractivation than with ADP alone.

Other functional assays include, for example, microphysiometer orbiosensor assays (see Hafner, 2000, Biosens. Bioelectron. 15: 149-158,incorporated herein by reference). The intracellular level of arachinoidacid can also be determined as described in Gijon et al., 2000, J. Biol.Chem., 275:20146-20156.

II. Diagnostic Assays Based Upon the Interaction of GPR86 and ADP:

Signaling through GPCRs is instrumental in the pathology of a largenumber of diseases and disorders. GPR86, which is expressed in cells ofthe lymphocyte lineages, platelets, spleen as well as leukemic cells,can have a role in immune processes, cancer, thrombosis and associateddisorders or diseases. The GPR86 expression pattern also includes thebrain and further suggests a potential role as an ADP neurotransmitter.

The expression pattern of GPR86 and the knowledge with respect todisorders generally mediated by GPCRs suggests that GPR86 can beinvolved in disturbances of cell migration, cancer, development oftumours and tumour metastasis, inflammatory and neo-plastic processes,wound and bone healing and dysfunction of regulatory growth functions,diabetes, obesity, anorexia, bulimia, acute heart failure, hypotension,hypertension, urinary retention, osteoporosis, angina pectoris,myocardial infarction, restenosis, atherosclerosis, thrombosis and othercardiovascular diseases, autoimmune and inflammatory diseases, diseasescharacterized by excessive smooth muscle cell proliferation, aneurysms,diseases characterized by loss of smooth muscle cells or reduced smoothmuscle cell proliferation, stroke, ischemia, ulcers, allergies, benignprostatic hypertrophy, migraine, vomiting, psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression,depression, delirium, dementia and severe mental retardation,degenerative diseases, neurodegenerative diseases such as Alzheimer'sdisease or Parkinson's disease, and dyskinasias, such as Huntington'sdisease or Gilles de la Tourett's syndrome and other related diseasesincluding thrombosis and other cardiovascular diseases, autoimmune andinflammatory diseases.

The interaction of GPR86 with ADP can be used as the basis of assays forthe diagnosis or monitoring of diseases, disorders or processesinvolving GPR86 signaling. Diagnostic assays for GPR86-related diseasesor disorders can have several different forms. First, diagnostic assayscan measure the amount of GPR86, genes or mRNA in a sample of tissue.Assays that measure the amount of mRNA encoding GPR86 polypeptide alsofit into this category. Second, assays can evaluate the qualities of thereceptor or the ligand. For example, assays that determine whether anindividual expresses a mutant or variant form of GPR86 or a polypeptideligand can be used diagnostically. Third, assays that measure one ormore activities of GPR86 polypeptide can be used diagnostically.

A. Assays that Measure the Amount of GPR86

GPR86 levels can be measured and compared to standards in order todetermine whether an abnormal level of the receptor or its ligand ispresent in a sample, either of which indicate probable dysregulation ofGPR86 signaling. Polypeptide levels are measured, for example, byimmunohistochemistry using antibodies specific for the polypeptide. Asample isolated from an individual suspected of suffering from a diseaseor disorder characterized by GPR86 activity is contacted with anantibody for GPR86, and binding of the antibody is measured as known inthe art (e.g., by measurement of the activity of an enzyme conjugated toa secondary antibody).

Another approach to the measurement of GPR86 levels uses flow cytometryanalysis of cells from an affected tissue. Methods of flow cytometry,including the fluorescent labeling of antibodies specific for GPR86, arewell known in the art. Other approaches include radioimmunoassay orELISA. Methods for each of these are also well known in the art.

The amount of binding detected is compared to the binding in a sample ofsimilar tissue from a healthy individual, or from a site on the affectedindividual that is not so affected. An increase of 10% or more relativeto the standard is diagnostic for a disease or disorder characterized byGPR86 dysregulation.

GPR86 expression can also be measured by determining the amount of mRNAencoding the polypeptides in a sample of tissue. Levels of mRNA can bemeasured by quantitative or semi-quantitative PCR. Methods of“quantitative” amplification are well known to those of skill in theart, and primer sequences for the amplification of both GPR86 aredisclosed herein. A common method of quantitative PCR involvessimultaneously co-amplifying a known quantity of a control sequenceusing the same primers. This provides an internal standard that can beused to calibrate the PCR reaction. Detailed protocols for quantitativePCR are provided in PCR Protocols, A Guide to Methods and Applications,Innis et al., Academic Press, Inc. N.Y., (1990), which is incorporatedherein by reference. An increase of 10% or more in the amount of mRNAencoding GPR86 in a sample, relative to the amount expressed in a sampleof like tissue from a healthy individual or in a sample of tissue froman unaffected location in an affected individual is diagnostic for adisease or disorder characterized by dysregulation of GPR86 signaling.

B. Qualitative Assays

Assays that evaluate whether or not the GPR86 polypeptide or the mRNAencoding it are wild-type or not can be used diagnostically. In order todiagnose a disease or disorder characterized by GPR86 dysregulation inthis manner, RNA isolated from a sample is used as a template for PCRamplification of GPR86. The amplified sequences are then either directlysequenced using standard methods, or are first cloned into a vector,followed by sequencing. A difference in the sequence that changes one ormore encoded amino acids relative to the sequence of wild-type GPR86 canbe diagnostic of a disease or disorder characterized by dysregulation ofGPR86 signaling. It can be useful, when a change in coding sequence isidentified in a sample, to express the variant receptor or ligand andcompare its activity to that of wild type GPR86. Among other benefits,this approach can provide novel mutants, including constitutively activeand null mutants.

In addition to standard sequencing methods, amplified sequences can beassayed for the presence of specific mutations using, for example,hybridization of molecular beacons that discriminate between wild typeand variant sequences. Hybridization assays that discriminate on thebasis of changes as small as one nucleotide are well known in the art.Alternatively, any of a number of “minisequencing” assays can beperformed, including, those described, for example, in U.S. Pat. Nos.5,888,819, 6,004,744 and 6,013,431 (incorporated herein by reference).These assays and others known in the art can determine the presence, ina given sample, of a nucleic acid with a known polymorphism.

If desired, array or microarray-based methods can be used to analyze theexpression or the presence of mutation, in GPR86 sequences. Array-basedmethods for minisequencing and for quantitation of nucleic acidexpression are well known in the art.

C. Functional Assays.

Diagnosis of a disease or disorder characterized by the dysregulation ofGPR86 signaling can also be performed using functional assays. To do so,cell membranes or cell extracts prepared from a tissue sample are usedin an assay of GPR86 activity as described herein (e.g., ligand bindingassays, the GTP-binding assay, GTPase assay, adenylate cyclase assay,cAMP assay, arachidonic acid level, phospholipid breakdown, diacylglycerol or inositol triphosphate assays, PKC activation assay, orkinase assay). The activity detected is compared to that in a standardsample taken from a healthy individual or from an unaffected site on theaffected individual. As an alternative, a sample or extract of a samplecan be applied to cells expressing GPR86, followed by measurement ofGPR86 signaling activity relative to a standard sample. A difference of10% or more in the activity measured in any of these assays, relative tothe activity of the standard, is diagnostic for a disease or disordercharacterized by dysregulation of GPR86 signaling.

Modulation of GPR86 Activity in a Cell According to the Invention

The discovery of ADP as a ligand of GPR86 provides methods of modulatingthe activity of a GPR86 polypeptide in a cell. GPR86 activity ismodulated in a cell by delivering to that cell an agent that modulatesthe function of a GPR86 polypeptide. This modulation can be performed incultured cells as part of an assay for the identification of additionalmodulating agents, or, for example, in an animal, including a human.Agents include ADP and its analogues as defined herein, as well asadditional modulators identified using the screening methods describedherein including but not limited to any of the ADP analogues presentedin U.S. Pat. No. 5,700,786.

An agent can be delivered to a cell by adding it to culture medium. Theamount to deliver will vary with the identity of the agent and with thepurpose for which it is delivered. For example, in a culture assay toidentify antagonists of GPR86 activity, one will preferably add anamount of ADP that half-maximally activates the receptors (e.g.,approximately EC₅₀), preferably without exceeding the dose required forreceptor saturation. This dose can be determined by titrating the amountof ADP to determine the point at which further addition of ADP has noadditional effect on GPR86 activity.

When a modulator of GPR86 activity is administered to an animal for thetreatment of a disease or disorder, the amount administered can beadjusted by one of skill in the art on the basis of the desired outcome.Successful treatment is achieved when one or more measurable aspects ofthe pathology (e.g., tumor cell growth, accumulation of inflammatorycells) is changed by at least 10% relative to the value for that aspectprior to treatment.

Candidate Modulators Useful According to the Invention

The invention provides for a compound that is a modulator of a receptorof the invention.

Preferably a candidate modulator is a nucleotide or a nucleotide whichbinds to a sugar, including but not limited to ADP-glucose orADP-galactose. A candidate modulator may also be any ADP analog known inthe art as well as any ligand that binds to the UDP glucose receptor.

The candidate compound may be a synthetic compound, or a mixture ofcompounds, or may be a natural product (e.g. a plant extract or culturesupernatant). A candidate compound according to the invention includes asmall molecule that can be synthesized, a natural extract, peptides,proteins, carbohydrates, lipids etc. . . .

Candidate modulator compounds from large libraries of synthetic ornatural compounds can be screened. Numerous means are currently used forrandom and directed synthesis of saccharide, peptide, and nucleic acidbased compounds. Synthetic compound libraries are commercially availablefrom a number of companies including Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemicallibrary is available from Aldrich (Milwaukee, Wis.). Combinatoriallibraries are available and can be prepared. Alternatively, libraries ofnatural compounds in the form of bacterial, fungal, plant and animalextracts are available from e.g., Pan Laboratories (Bothell, Wash.) orMycoSearch (NC), or are readily produceable by methods well known in theart. Additionally, natural and synthetically produced libraries andcompounds are readily modified through conventional chemical, physical,and biochemical means.

Useful compounds may be found within numerous chemical classes. Usefulcompounds may be organic compounds, or small organic compounds. Smallorganic compounds have a molecular weight of more than 50 yet less thanabout 2,500 daltons, preferably less than about 750, more preferablyless than about 350 daltons. Exemplary classes include heterocycles,peptides, saccharides, steroids, and the like. The compounds may bemodified to enhance efficacy, stability, pharmaceutical compatibility,and the like. Structural identification of an agent may be used toidentify, generate, or screen additional agents. For example, wherepeptide agents are identified, they may be modified in a variety of waysto enhance their stability, such as using an unnatural amino acid, suchas a D-amino acid, particularly D-alanine, by functionalizing the aminoor carboxylic terminus, e.g. for the amino group, acylation oralkylation, and for the carboxyl group, esterification or amidification,or the like.

For primary screening, a useful concentration of a candidate compoundaccording to the invention is from about 1 μM to about 60 μM or more(i.e., 100 μM, 1 mM, 10 mM, 100 mM, 1M etc . . . ). The primaryscreening concentration will be used as an upper limit, along with nineadditional concentrations, wherein the additional concentrations aredetermined by reducing the primary screening concentration at half-logintervals (e.g. for 9 more concentrations) for secondary screens or forgenerating concentration curves.

Antibodies Useful According to the Invention

The invention provides for antibodies to GPR86. Antibodies can be madeusing standard protocols known in the art (See, for example, Antibodies:A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988). A mammal, such as a mouse, hamster, or rabbit can be immunizedwith an immunogenic form of the peptide (e.g., GPR86 polypeptide or anantigenic fragment which is capable of eliciting an antibody response,or a fusion protein as described herein above). Immunogens for raisingantibodies are prepared by mixing the polypeptides (e.g., isolatedrecombinant polypeptides or synthetic peptides) with adjuvants.Alternatively, GPR86 polypeptides or peptides are made as fusionproteins to larger immunogenic proteins. Polypeptides can also becovalently linked to other larger immunogenic proteins, such as keyholelimpet hemocyanin. Alternatively, plasmid or viral vectors encodingGPR86 polypeptide, or a fragment of these proteins, can be used toexpress the polypeptides and generate an immune response in an animal asdescribed in Costagliola et al., 2000, J. Clin. Invest. 105:803-811,which is incorporated herein by reference. In order to raise antibodies,immunogens are typically administered intradermally, subcutaneously, orintramuscularly to experimental animals such as rabbits, sheep, andmice. In addition to the antibodies discussed above, geneticallyengineered antibody derivatives can be made, such as single chainantibodies.

The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA, flow cytometry or otherimmunoassays can also be used with the immunogen as antigen to assessthe levels of antibodies. Antibody preparations can be simply serum froman immunized animal, or if desired, polyclonal antibodies can beisolated from the serum by, for example, affinity chromatography usingimmobilized immunogen.

To produce monoclonal antibodies, antibody-producing splenocytes can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with GPR86 polypeptide,and monoclonal antibodies isolated from the media of a culturecomprising such hybridoma cells.

High Throughput Screening Kit

A high throughput screening kit according to the invention comprises allthe necessary means and media for performing the detection of amodulator compound including an agonist, antagonist, inverse agonist orinhibitor to the receptor of the invention in the presence of ADP,preferably at a concentration in the range of 1 nM to 10 μM. The kitcomprises the following successive steps. Recombinant cells of theinvention, comprising and expressing the nucleotide sequence encodingthe GPR86 (P2Y₁₃) receptor, are grown on a solid support, such as amicrotiter plate, more preferably a 96 well microtiter plate, accordingto methods well known to the person skilled in the art especially asdescribed in WO 00/02045. Modulator compounds according to theinvention, at concentrations from about 1 nM to 10 μM or more, are addedto the culture media of defined wells in the presence of an appropriateconcentration of ADP (preferably in the range of 1 nM to 1 μM).

Secondary messenger assays, amenable to high throughput screeninganalysis, are performed including but not limited to the measurement ofintracellular levels of cAMP, intracellular inositol phosphate,intracellular diacylglycerol concentrations, arachinoid acidconcentration or MAP kinase or tyrosine kinase activity (as decribedabove). For example, the GPR86 activity, as measured in a cyclic AMPassay, is quantified by a radioimmunoassay as previously described (26).Results are compared to the baseline level of GPR86 activity obtainedfrom recombinant cells according to the invention in the presence of ADPbut in the absence of added modulator compound. Wells showing at least 2fold, preferably 5 fold, more preferably 10 fold and most preferably a100 fold or more increase or decrease in GPR86 activity as compared tothe level of activity in the absence of modulator, are selected forfurther analysis.

Other Kits Useful According to the Invention

The invention provides for kits useful for screening for modulators ofGPR86 activity, as well as kits useful for diagnosis of diseases ordisorders characterized by dysregulation of GPR86 signaling. Kits usefulaccording to the invention can include an isolated GPR86 polypeptide(including a membrane- or cell-associated GPR86 polypeptide, e.g., onisolated membranes, cells expressing GPR86, or, on an SPR chip). A kitcan also comprise an antibody specific for GPR86. Alternatively, or inaddition, a kit can contain cells transformed to express GPR86polypeptide. In a further embodiment, a kit according to the inventioncan contain a polynucleotide encoding a GPR86 polypeptide. In a stillfurther embodiment, a kit according to the invention may comprise thespecific primers useful for amplification of GPR86 as described below.All kits according to the invention will comprise the stated items orcombinations of items and packaging materials therefore. Kits will alsoinclude instructions for use.

Transgenic Animals

Transgenic mice provide a useful tool for genetic and developmentalbiology studies and for the determination of the function of a novelsequence. According to the method of conventional transgenesis,additional copies of normal or modified genes are injected into the malepronucleus of the zygote and become integrated into the genomic DNA ofthe recipient mouse. The transgene is transmitted in a Mendelian mannerin established transgenic strains. Constructs useful for creatingtransgenic animals comprise genes under the control of either theirnormal promoters or an inducible promoter, reporter genes under thecontrol of promoters to be analyzed with respect to their patterns oftissue expression and regulation, and constructs containing dominantmutations, mutant promoters, and artificial fusion genes to be studiedwith regard to their specific developmental outcome. Typically, DNAfragments on the order of 10 kilobases or less are used to construct atransgenic animal (Reeves, 1998, New. Anat., 253:19). Transgenic animalscan be created with a construct comprising a candidate gene containingone or more polymorphisms according to the invention. Alternatively, atransgenic animal expressing a candidate gene containing a singlepolymorphism can be crossed to a second transgenic animal expressing acandidate gene containing a different polymorphism and the combinedeffects of the two polymorphisms can be studied in the offspringanimals.

Other Transgenic Animals

The invention provides for transgenic animals that include but are notlimited to transgenic mice, rabbits, rats, pigs, sheep, horses, cows,goats, etc. A protocol for the production of a transgenic pig can befound in White and Yannoutsos, Current Topics in Complement Research:64^(th) Forum in Immunology, pp. 88-94; U.S. Pat. No. 5,523,226; U.S.Pat. No. 5,573,933: PCT Application WO93/25071; and PCT ApplicationWO95/04744. A protocol for the production of a transgenic mouse can befound in U.S. Pat. No. 5,530,177. A protocol for the production of atransgenic rat can be found in Bader and Ganten, Clinical andExperimental Pharmacology and Physiology, Supp. 3:S81-S87, 1996. Aprotocol for the production of a transgenic cow can be found inTransgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert,Academic Press, Inc. A protocol for the production of a transgenicrabbit can be found in Hammer et al., Nature 315:680-683, 1985 andTaylor and Fan, Frontiers in Bioscience 2:d298-308, 1997.

Knock Out Animals

i. Standard

Knock out animals are produced by the method of creating gene deletionswith homologous recombination. This technique is based on thedevelopment of embryonic stem (ES) cells that are derived from embryos,are maintained in culture and have the capacity to participate in thedevelopment of every tissue in the mouse when introduced into a hostblastocyst. A knock out animal is produced by directing homologousrecombination to a specific target gene in the ES cells, therebyproducing a null allele of the gene. The potential phenotypicconsequences of this null allele (either in heterozygous or homozygousoffspring) can be analyzed (Reeves, supra).

ii. In Vivo Tissue Specific Knock Out in Mice Using Cre-lox.

The method of targeted homologous recombination has been improved by thedevelopment of a system for site-specific recombination based on thebacteriophage P1 site specific recombinase Cre. The Cre-loxPsite-specific DNA recombinase from bacteriophage P1 is used intransgenic mouse assays in order to create gene knockouts restricted todefined tissues or developmental stages. Regionally restricted geneticdeletion, as opposed to global gene knockout, has the advantage that aphenotype can be attributed to a particular cell/tissue (Marth, 1996,Clin. Invest. 97: 1999). In the Cre-loxP system one transgenic mousestrain is engineered such that loxP sites flank one or more exons of thegene of interest. Homozygotes for this so called ‘floxed gene’ arecrossed with a second transgenic mouse that expresses the Cre gene undercontrol of a cell/tissue type transcriptional promoter. Cre protein thenexcises DNA between loxP recognition sequences and effectively removestarget gene function (Sauer, 1998, Methods, 14:381). There are now manyin vivo examples of this method, including the inducible inactivation ofmammary tissue specific genes (Wagner et al., 1997, Nucleic Acids Res.,25:4323).

iii. Bac Rescue of Knock Out Phenotype

In order to verify that a particular genetic polymorphism/mutation isresponsible for altered protein function in vivo one can “rescue” thealtered protein function by introducing a wild-type copy of the gene inquestion. In vivo complementation with bacterial artificial chromosome(BAC) clones expressed in transgenic mice can be used for thesepurposes. This method has been used for the identification of the mousecircadian Clock gene (Antoch et al., 1997, Cell 89: 655).

Materials

Trypsin was from Flow Laboratories (Bioggio, Switzerland). Culturemedia, G418, fetal bovine serum (FBS), restriction enzymes, Platinum Pfxand Taq DNA polymerases were purchased from Life Technologies, Inc.(Merelbeke, Belgium). The radioactive product myo-D-[2-³H]inositol (17.7Ci/mmol) was from Amersham (Ghent, Belgium). Dowex AG1X8 (formate form)was from Bio-Rad Laboratories (Richmond, Calif.). ATP, ADP, adenosine,ADPβS (adenosine 5′-O-(2-thiodiphosphate)), A2P5P (adenosine2′,5′-diphosphate), A3P5P (adenosine 3′,5′-diphosphate), A3P5PS(adenosine 3′-phosphate 5′-phosphosulfate), UTP, UDP, ITP, IDP,UDP-glucose and 3-isobutyl-1-methyl-xanthine (IBMX) were obtained fromSigma Chemical Co. (St. Louis, Mo.). 2-methylthio-ADP (2MeSADP) and2-methylthio-ATP (2MeSATP) were from Research Biochemicals International(Natick, Mass.). Forskolin was purchased from Calbiochem. (Bierges,Belgium). Rolipram was a gift from the Laboratories Jacques Logeais(Trappes, France). pEFIN5 is an expression vector developed byEuroscreen (Brussels, Belgium). Monoclonal antibody specific for thedually phosphorylated forms of Erk1 and Erk2 (at Thr²⁰² and Tyr²⁰⁴) wasobtained from New England Biolabs (Beverly, Mass.).

Dosage and Mode of Administration

By way of example, a patient can be treated as follows by theadministration of a modulator of GPR86 (for example, an agonist,antagonist or inhibitor of GPR86, of the invention). A modulator ofGPR86 the invention can be administered to the patient, preferably in abiologically compatible solution or a pharmaceutically acceptabledelivery vehicle, by ingestion, injection, inhalation or any number ofother methods. The dosages administered will vary from patient topatient; a “therapeutically effective dose” can be determined, forexample but not limited to, by the level of enhancement of function(e.g., as determined in a second messenger assay described herein).Monitoring ADP binding will also enable one skilled in the art to selectand adjust the dosages administered. The dosage of a modulator of GPR86of the invention may be repeated daily, weekly, monthly, yearly, or asconsidered appropriate by the treating physician.

Pharmaceutical Compositions

The invention provides for compositions comprising a GPR86 modulatoraccording to the invention admixed with a physiologically compatiblecarrier. As used herein, “physiologically compatible carrier” refers toa physiologically acceptable diluent such as water, phosphate bufferedsaline, or saline, and further may include an adjuvant. Adjuvants suchas incomplete Freund's adjuvant, aluminium phosphate, aluminiumhydroxide, or alum are materials well known in the art.

The invention also provides for pharmaceutical compositions. In additionto the active ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carrier preparations which can beused pharmaceutically.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer' solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

For nasal administration, penetrants appropriate to the particularbarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner known in the art, e.g. by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. . . . Saltstend to be more soluble in aqueous or other protonic solvents that arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2%sucrose, 2%-7% mannitol at a Ph range of 4.5 to 5.5 that is combinedwith buffer prior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in a acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition with information including amount, frequency andmethod of administration.

EXAMPLES

The invention is illustrated by the following nonlimiting exampleswherein the following materials and methods are employed. The entiredisclosure of each of the literature references cited hereinafter areincorporated by reference herein.

Example 1

Cloning and Sequencing

An intronless coding sequence encoding a novel receptor strongly relatedto the human P2Y₁₂ receptor was identified on the genomic cloneRP11-25K24 (GenBank accession AC024886) located in the 3q24 region andin the following patent: WO 00/31258; ARENA; sequence number 18.

Specific oligonucleotide primers were synthesized on the basis of thesequence of the GPR86 human receptor: a sense primer5′-CCGGAATTCACCATGAACACCACAGTGATGC-3′ (SEQ ID NO: 4) and an antisenseprimer 5′-CTTGTCTAGATCAGCCTAAGGTTATGTTGTC-3′ (SEQ ID NO: 5). Apolymerase chain reaction (PCR) was performed on three different spleencDNAs using the Platinum Pfx DNA Polymerase. The amplificationconditions were as follows: 94° C., 15 s; 50° C., 30 s; 68° C., 2 minfor 35 cycles. Amplifications resulted in a fragment of 1 kilobasecontaining the entire coding sequence of the GPR86 gene. The codingsequence was then subcloned between the EcoRI and XbaI sites of thebicistronic pEFIN5 expression vector and sequenced on both strands foreach of the three cDNAs using the BigDye Terminator cycle sequencing kit(Applied Biosystems, Warrington, Great Britain).

This 1002 base pairs (bp)-open reading frame was also identifiedrecently by Wittenberger et al. (GenBank accession AF295368) andreported to encode an orphan G-protein-coupled receptor that they calledGPR86 (24). The start codon is preceded by a stop codon 18 bp upstream.Oligonucleotide primers were synthesized on the basis of this codingsequence. They were used in PCR starting from spleen cDNA. A PCR productwith a size compatible with GPR86 coding sequence was inserted into thepEFIN5 expression vector and sequenced on both strands (FIG. 1). Theputative membrane-spanning domains are underlined and numbered I to VII.The putative sites of phosphorylation by protein kinase A or by proteinkinase C are indicated respectively by a black circle (●) or a blackdiamond (♦). The potential N-glycosylation sites are indicated by ablack square (▪).

The sequence obtained matched perfectly to the sequence of Wittenbergeret al. amplified from human cDNA libraries from fetal brain andplacenta. The 1002 bp-open reading frame starts with an ATG-codon in aKozak consensus and encodes a protein of 333 amino acids. The peptidicsequence contains three potential sites for N-linked glycosylation (twoin the extracellular N-terminal part (N² and N¹⁰) and one in the thirdextracellular loop (N²⁶⁴), two potential sites for phosphorylation byprotein kinase C (one in the third intracellular loop (S²¹⁷) and one inthe carboxyterminal part (T³⁰⁴)) and one by protein kinase A (in thecarboxyterminal part (T³¹⁶)) (FIG. 1). The novel receptor displays asignificant homology with the human P2Y₁₂ and UDP-glucose receptors(FIG. 2), 48% and 45% amino acid identity respectively. The similaritywith the other P2Y receptors is much lower (FIG. 2), for example, 25%and 26% amino acid identity with respectively the human P2Y₁ and P2Y₂receptors. Alignment of the amino acid sequence of GPR86 (P2Y₁₃) withpurinergic receptors (P2Y1, -2, -4, -6, -11, -12), UDP glucose receptorand other purinergic related sequences (GPR17, GPR87, H963) wereperformed using ClustalX algorithm. Then, the dendrogram was constructedusing TreeView algorithm.

Example 2

Tissue Distribution of GPR86 Human Receptor

GPR86 mRNA was amplified by RT-PCR in several human tissues (FIG. 3A).

Reverse transcription-polymerase chain reaction (RT-PCR) experimentswere carried out using a panel of polyA⁺ RNA (Clontech). The GPR86primers were as follows: GPR86 sense primer (5′-TGTGTCGTTTTTCTTCGGTG-3′)(SEQ ID NO: 6) and GPR86 antisense primer (5′-CTGCCAAAAAGAGAGTTG-3′)(SEQ ID NO: 7). The expected size of the amplified DNA band was 575 bp.Two primers synthesized on the basis of aldolase coding sequence wereused as controls to produce a product with an expected size of 443 bp:aldolase sense primer 5′-GGCAAGGGCATCCTGGCTGC-3′ (SEQ ID NO: 8) andaldolase antisense reverse 5′-TAACGGGCCAGAACATTGGCATT-3′ (SEQ ID NO: 9).Approximately 75 ng of poly A⁺ RNA was reverse transcribed withSuperscript II (Life Technologies, Inc., Merelbeke, Belgium) and usedfor PCR. PCR was performed using the Taq polymerase under the followingconditions: denaturation at 94° C. for 3 min, 38 cycles at 94° C. for 1min, 58° C. for 2 min and 72° C. for 2 min. Aliquots (10 μl) of the PCRreaction were analysed by 1% agarose gel electrophoresis.

RT-PCR experiments were carried out using a panel of polyA⁺ RNA(Clontech) and specific primers of GPR86 sequence. The expected size ofthe amplified GPR86 and aldolase bands were respectively 575 and 443 bp.cDNA (−) indicates the negative control of the PCR reaction without cDNAtemplate. Aliquots (10 μl) of the PCR reaction were analysed by 1%agarose gel electrophoresis. A strong band of the expected size (575 bp)was detected in spleen and brain (adult), and at lower intensity inplacenta, lung, liver, spinal cord, thymus, small intestine, uterus,stomach, testis, fetal brain, and adrenal gland, but not in pancreas,heart, kidney, skeletal muscle, ovary, fetal aorta or the negativecontrol without cDNA (FIG. 3A). A 575 bp-band was also clearly detectedin lymph node and bone marrow, and weakly detected in peripheral bloodmononuclear cells (PBMC) (FIG. 3A). No signal was detected in peripheralblood lymphocytes (PBL) and polymorphonuclear cells (PMN) (FIG. 3A).GPR86 messengers were detected in different brain regions (thalamus,caudate nucleus, substantia nigra, hippocampus, cerebellum, corpuscallosum and amygdala) (FIG. 3B). The amplification of a fragment ofaldolase coding sequence was used as control.

Northern blot analysis with hGPR86 revealed a strong 2.9 kb transcriptin spleen and a weaker one in liver, placenta, leukocytes, and brain.Evaluation of the expression of hGPR86 in different brain regionsrevealed the 2.9 kb transcript as a strong signal in substantia nigra,thalamus, and medulla, less strong in frontal and temporal lobe,putamen, amygdala, caudate nucleus, hippocampus, spinal cord, corpuscallosum, and weak in cerebellum and occipital lobe. The transcript wasnot detectable in the cerebral cortex. The wide spread expression ofhGPR86 shown in the Northern blot analysis is reflected by the origin of16 EST sequences found for this GPCR in the public database, derivedfrom diverse tissues as germ cell tumors, fetal liver, fetal spleen,colon, pregnant uterus and multiple sclerosis lesions. The PCRamplification of hGPR86 from brain and placenta cDNAs is also inagreement with these results (24).

Example 3

Stable Expression of the Novel Receptor in 1321N1 Astrocytoma Cells

The complete sequence of the novel receptor was introduced in the pEFIN5expression vector in order to transfect the 1321N1 astrocytoma cellline, used previously to characterize several P2Y subtypes (5, 13, 14).1321N1 astrocytoma cells expressing G□₁₆ protein were transfected withthe recombinant GPR86-pEFIN5 plasmid or with the plasmid alone.

CHO-K1 and 1321N1 cells were transfected with the recombinantGPR86-pEFIN5 plasmid or with the plasmid alone using the FuGENE™6transfection reagent (Roche Molecular Biochemicals). A clone called AG32corresponding to 1321N1 cells previously transfected with pERAEQ2plasmid encoding Gα₁₆ (provided by Euroscreen), was transfected. TheCHO-K1 and 1321N1 transfected cells were selected with 400 μg/ml G418 incomplete medium (10% FBS, 100 units/ml penicillin, 100 μg/mlstreptomycin and 2.5 μg/ml amphotericin B in respectively Ham's F12 orDMEM (Dulbecco's modified Eagle's) medium) two days after transfectionand maintained in the same medium. The AG32 cells were maintained in thesame DMEM complete medium supplemented with 500 μg/ml zeocin.

The pool of G418-resistant clones was tested for its functional IP₃response to several nucleotides, according to the method describedabove. The cells were challenged by various nucleotides at aconcentration of 100 μM for 30 s: ATP, ADP, UTP, UDP, ITP, IDP, TDP. Noresponse was obtained in 1321N1 cells expressing GPR86 receptor alone,while a strong IP₃ response to histamine was observed in these cells,but ADP, UDP and IDP induced an IP₃ response in 1321N1 cells expressingboth GPR86 receptor and Gα₁₆ protein. No IP₃ response was observed forthe other nucleotides, except for ATP and 2MeSATP, but these responseswere lost after HPLC-purification (data not shown). No IP₃ response wasobserved in response to any nucleotide in 1321N1 or 1321N1-Gα₁₆ cellstransfected with the wild-type pEFIN5 vector and used as negativecontrol.

In 1321N1 cells expressing both GPR86 receptor and the Gα₁₆,concentration-action curves were established for ADP, IDP and UDP andrevealed the strong affinity of GPR86 for ADP. The following range ofpotency was obtained: ADP>>>IDP>UDP. The affinity of GPR86 for ADP wasapproximately one thousand-fold greater than that of IDP and UDP.Concentration-action curves for ADP, 2MeSADP and ADPβS. The followingEC₅₀ values were computed respectively for ADP, 2MeSADP and ADPβS wereobtained: 11.4±2.2 nM, 14.2±3.0 nM and 48.4±0.4 nM (mean±S.D. of threeindependent experiments) (FIG. 4A). 1321N1 transfected cells wereincubated in the presence of various concentrations of ADP, 2MeSADP andADPβS for 30 s. The data represent the mean±S.D. of triplicateexperimental points obtained in one representative experiment of three.No IP₃ response was obtained for A2P5P (ADP 2′,5′-diphosphate), A3P5P(adenosine 3′,5′-diphosphate) and A3P5PS (adenosine 3′-phosphate5′-phosphosulfate).

Effects of 2MeSATP and ATP were obtained at concentrations higher thanfor the respective diphosphates nucleotides (FIG. 4B). 1321N1transfected cells were pre-incubated with or without 100 ng/ml pertussistoxin for 18 h and then incubated in the presence of ADP (300 nM) orwater (CONT) for 30 s. The data represent the mean±S.D. of triplicateexperimental points obtained in one representative experiment of two.

As discussed previously, commercial nucleotide powders are contaminatedby degradation products (4, 13, 28). Contamination is usually 1% for ATPand about 10% for 2MeSATP (28). 2MeSATP and ATP solutions (1 mM) weretreated at room temperature with 20 units/ml CPK and 10 mM CP during 90min. This ATP-regenerating system circumvents problems arising from thecontamination and degradation of triphosphate nucleotide solutions (28).In these conditions, the responses to ATP and 2MeSATP were abolished(FIG. 4B).

An inhibition of 86±8% (mean±range of two independent experiments) ofthe ADP response (300 nM) after a 18 hours pretreatment of thetransfected cells with 100 ng/ml pertussis toxin (PTx) was observed(FIG. 4C). 1321N1 transfected cells were pre-incubated with or without100 ng/ml pertussis toxin (PTx) for 18 h and then incubated in thepresence of ADP (300 nM) or control medium (CONT) for 30 s. The datarepresent the mean±S.D. of triplicate experimental points obtained inone representative experiment of two.

Example 4

Stable Expression of the Novel Receptor in CHO-K1 Cells

The potential effect of nucleotides was tested on the cAMP pathway inCHO-K1 cells expressing the human GPR86 receptor. Significantinhibitions of the cAMP level were observed at low concentrations of ADP(FIG. 5A) and 2MeSADP in the presence of forskolin (4 μM). CHO-K1transfected cells were incubated in the presence of variousconcentrations of ADP and 4 μM forskolin for 10 min. The data representthe mean±S.D. of triplicate experimental points obtained in onerepresentative experiment of three and 2MeSADP in the presence offorskolin 4 μM. The IC₅₀ of ADP was 1.5±0.6 nM (mean±S.D. of threeindependent experiments) with a maximal inhibition percentage of 52±7%at 30 nM (mean±S.D. of three independent experiments). A second phasewas observed at concentrations higher than 30 nM: the inhibition ofadenylyl cyclase decreased and a small increase was observed at 10 μM(FIG. 5A). After an 18 h-pretreatment of the transfected CHO-K1 cellswith 100 ng/ml pertussis toxin, ADP (100 nM and 100 μM) inducedsignificant increases of the cAMP level (FIG. 5B). CHO-K1 transfectedcells were incubated in the presence of various concentrations of ADP an4 μM forskolin for 10 minutes. The data represent the mean+/−S.D. oftriplicate experimental points obtained in one representative experimentof three. CHO-K1 transfected cells were pre-incubated with or without100 ng/ml pertussis toxin for 18 h and then incubated in the presence ofADP (100 nM and 100 μM) or water (CONT) and 4 μM forskolin for 10 min.The data represent the mean±S.D. of triplicate experimental pointsobtained in one representative experiment of two. The biphasic effect ofADP on adenylyl cyclase has been reproduced in 1321N1 cells transfectedwith the human GPR86 receptor.

To investigate changes in the activation status of the MAP kinases Erk1and Erk2 upon stimulation of the human GPR86 receptor, whole CHO-K1transfected cell extracts were analysed by Western blotting using aspecific antibody for the dually phosphorylated kinases (at Thr²⁰² andTyr²⁰⁴), which are the active forms of Erk. Western Blot Analysis ofphosphorylated Erk1 and Erk2 proteins.

GPR86-transfected CHO-K1 cells were seeded at 1.5×10⁶ cells/dish. After24 h, the cells were serum-starved for 2 h in KRH buffer. Afterstimulation with the agonist, the cells were scraped in 1 ml of PBS pH7.3 (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄.7H₂O and 1.4 mM KH₂PO₄).The cells were recovered by centrifugation and lysed in 150 μl ofLaemmli buffer (10% (w/v) glycerol, 5% (v/v) mercaptoethanol, 2.3% (w/v)SDS, 62.5 mM Tris-HCl pH 6.8). The protein concentration was determinedusing the method of Minamide and Bamburg (27). The same amount ofprotein for each condition was electrophoresed in a 12%SDS-polyacrylamide gel. Proteins were then transferred overnight at 60 Vand 4° C. onto a nitrocellulose membrane using 20 mM Tris, 154 mMglycine, 20% (v/v) methanol as a transfer buffer. Immunodetection wasachieved using the enhanced chemiluminescence Western blotting detectionsystem (ECL, Amersham Phammacia Biotech) using a biotinylated-secondarymouse antibody ( 1/25,000). The monoclonal antibody specific for thedually phosphorylated forms of Erk1 and Erk2 (at Thr²⁰² and Tyr²⁰⁴) wasused at 1/1000-dilution.

In CHO-K1 cells, phosphorylated Erk1 and Erk2 had the predictedmolecular mass of 44 and 42 kDa, respectively. Stimulation of the humanGPR86 receptor stably expressed in CHO-K1 cells with 1 μM ADP or2-MeSADP led to a strong phosphorylation of Erk2 (FIG. 6A).GPR86-transfected CHO-K1 cells were stimulated during different times orwith various concentrations of ADP and 2MeSADP or water (CONT). Theeffect of 100 ng/ml pertussis toxin (PTx) has been tested at 5 and 30min in the presence of 1 μM ADP or 2MeSADP. Blotting and immunodetectionwere achieved as described using the enhanced chemiluminescence Westernblotting detection system (ECL, Amersham Pharmacia Biotech).

Erk1 was also weakly phosphorylated. Erk phosphorylation was detectedafter 1 min of stimulation and increased with time (FIG. 6A). Themaximal response was obtained after 5 min of stimulation with ADP orwith 2-MeSADP, after which Erk activation slowly decreased to the basallevel after 1 h. To determine if endogenous receptors can be responsiblefor ADP or 2-MeSADP-dependent Erk activation in CHO-K1 cells, thesenucleotides were tested on cells transfected with the empty pEFIN5vector: Erk1 or Erk2 phosphorylation were not observed when CHO-K1control cells were incubated 5 min, 20 min or 1 h with 1 μM of 2-MeSADPor ADP. The concentration-dependence of the Erk phosphorylation inducedby ADP was determined at the peak of the transient response (5 min).Stimulation of the human GPR86 receptor with ADP or 2MeSADP (1 nM to 10μM) led to a concentration-dependent phosphorylation of Erk1 and Erk2(FIG. 6B). A maximal effect was obtained at 1 μM, but a significanteffect was already observed at 10 nM for both agonists. In order toevaluate the involvement of G_(i) protein in these effects, wepre-incubated GPR86—CHO-K1 transfected cells with pertussis toxin (100ng/ml for 18 h) prior to the ADP (1 μM) or the 2-MeSADP (1 μM)stimulation. The Erk phosphorylation normally induced by 5 or 30 minutesof ADP or 2-MeSADP treatment was strongly inhibited (FIG. 6B).

The cloning and pharmacological characterization of a novel humanG-protein-coupled receptor of the P2Y family, tentatively called P2Y₁₃corresponds to the previously described GPR86 orphan receptor (24).Concerning its sequence, the homology with the P2Y₁ to P2Y₁₁ subtypes isrestricted to around 25%. On the contrary, the GPR86 (P2Y₁₃) receptordisplays a significant homology with the human P2Y₁₂ and UDP-glucosereceptors. The closest G-coupled receptor is the human P2Y₁₂ receptor(48% amino acid identity) which is also a receptor responsive to ADP.Mutagenesis experiments with the P2Y₂ receptor have identified threepositively charged amino acids in the sixth and seventh transmembranedomains (His²⁶², Arg²⁶⁵ and Arg²⁹²), which play a crucial role innucleotide binding (presumably by neutralizing the negative charge ofthe phosphate groups) (28). The first two residues are conserved in theGPR86 (P2Y₁₃) receptor respectively at positions 251 and 254. These tworesidues are also conserved in the P2Y₁₂ and UDP-glucose receptors. TheArg²⁹² residue of the P2Y₂ receptor is replaced by a negatively chargedglutamate residue in P2Y₁₂, GPR86 (P2Y₁₃) and UDP-glucose receptors andcould have a great importance for the pharmacology of the receptor.

P2Y₁₂ and GPR86 (P2Y₁₃) receptors thus form a subgroup of P2Y receptorsstructurally different from the other P2Y receptors and which share ahigh affinity for their ligand, ADP. The EC₅₀ value of ADP for the GPR86(P2Y₁₃) receptor is 20 to 1000-fold lower than that of the respectiveligands of other P2Y receptors in comparable transfected cell lines.From a pharmacological point of view, the relative affinities of ADP and2MeSADP allow one to discriminate between the P2Y₁₂ and GPR86 (P2Y₁₃)subtypes. Similar affinities were observed for the GPR86 (P2Y₁₃)receptor, whereas 2MeSADP displays a 10 to 100-fold higher potency thanADP for the P2Y₁₂ receptor, depending on the expression system (21, 22).

The presence of the Gα₁₆ protein was necessary to couple the GPR86(P2Y₁₃) receptor to phospholipase C in 1321N1 cells. The stronginhibitory effect of pertussis toxin on the IP₃ accumulation induced byADP in 1321N1-Gα₁₆ transfected cells suggests a synergism between Gα₁₆and G_(i) proteins. Such a phenomenon has been described previously inHEL cells, where Ca²⁺ mobilisation by P2Y₂ agonists is inhibitedcompletely by Gα₁₆ antisense and partially by pertussis toxin (29).

Inhibition of adenylyl cyclase and the stimulation of MAP-kinases (ERK-1and 2) are transduction mechanisms associated to the GPR86 (P2Y₁₃)receptor and involving both G_(i) proteins. The biphasic effect of ADPon adenylyl cyclase is reminiscent of what has been observed for otherreceptors like the α₂ adrenergic receptor (30, 31). At lowconcentrations of agonist, there was an inhibition of adenylyl cyclasewhereas an increase was observed at higher concentrations, suggestingthe simultaneous coupling to two G-proteins with opposing effects. Thissimultaneous coupling could be an artefact of surexpression.

Concerning the tissue distribution of the human GPR86 (P2Y₁₃) receptor,a good correlation has been obtained between the present RT-PCR data andthe Northern blotting data obtained by Wittenberger et al. (24). Thehuman GPR86 (P2Y₁₃) receptor is especially expressed in human spleen andbrain, where it displays a large expression in different brain regions.The P2Y₁₂ receptor is also detected in the human brain and presents aglial expression pattern. Inhibition of cAMP formation by ADP has beendescribed in rat C6 glioma cells (32) and rat brain capillaryendothelial cells (33). In both models 2MeSADP was much more potent thanADP and 2MeSATP had a similar potency to 2MeSADP: these features suggestthe involvement of P2Y₁₂ rather than GPR86 (P2Y₁₃) receptors. Expressionof the GPR86 (P2Y₁₃) receptor in spleen, lymph nodes and bone marrowsuggests that it might play a role in hematopoiesis and the immunesystem.

This invention relates to the use of a human G protein-coupled receptor,GPR86 (P2Y₁₃), as a screening tool to identify agonists or antagonistsof the aequorin luminescence resulting from expression of this receptor.

The present invention will be described in more details in the followingexamples in reference to the enclosed figures.

Example 5

Production of a Transgenic Animal

Methods for generating non-human transgenic animals are describedherein. DNA constructs can be introduced into the germ line of a mammalto make a transgenic mammal. For example, one or several copies of theconstruct can be incorporated into the genome of a mammalian embryo bystandard transgenic techniques.

In an exemplary embodiment, a transgenic non-human animal is produced byintroducing a transgene into the germline of the non-human animal.Transgenes can be introduced into embryonal target cells at variousdevelopmental stages. Different methods are used depending on the stageof development of the embryonal target cell. The specific line(s) of anyanimal used should, if possible, be selected for general good health,good embryo yields, good pronuclear visibility in the embryo, and goodreproductive fitness.

Introduction of the P2Y₁₃ Receptor protein transgene into the embryo isaccomplished by any of a variety of means known in the art such asmicroinjection, electroporation, or lipofection. For example, an P2Y₁₃Receptor protein transgene is introduced into a mammal by microinjectionof the construct into the pronuclei of the fertilized mammalian egg(s)to cause one or more copies of the construct to be retained in the cellsof the developing mammal(s). Following introduction of the transgeneconstruct into the fertilized egg, the egg is incubated in vitro forvarying amounts of time, or reimplanted into the surrogate host, orboth. One common method is to incubate the embryos in vitro for about1-7 days, depending on the species, and then reimplant them into thesurrogate host.

The progeny of the transgenically manipulated embryos are tested for thepresence of the construct by Southern blot analysis of a segment oftissue. An embryo having one or more copies of the exogenous clonedconstruct stably integrated into the genome is used to establish apermanent transgenic mammal line carrying the transgenically introducedconstruct.

Litters of transgenically altered mammals are assayed after birth forthe incorporation of the construct into the genome of the offspring.This is done by hybridizing a probe corresponding to the DNA sequencecoding for the fusion protein or a segment thereof onto chromosomalmaterial from the progeny. Those mammalian progeny found to contain atleast one copy of the construct in their genome are grown to maturity.The transgenic mammals are bred to produce other transgenic progeny.

Transgenic females are tested for protein expression using an art-knownassay technique, e.g. a Western blot or enzymatic assay.

Example 6

GPR86 Activity

The activity of GPR 86 can be detected or measured as follows:Recombinant mammalian cells, for example 132N1 astrocytoma or CHO-K1cells, stably transfected with a suitable GPR86 (P2Y₁₃) expressionvector, are plated out onto tissue culture plates as described inexamples 3 and 4. At the appropriate cell density, usually between50-75% confluency, the culture media is replaced with a KRH buffersolution (Krebs-Ringer Hepes: 124 mM NaCL, 5 mM KCl, 1.25 mM MgSO₄, 1.45mM CaCl₂, 1.25 mM KH₂PO₄, 25 mM Hepes pH: 7.4 and 8 mM glucose)containing ADP ligand (preferably in the range of 1 nM to 1 μM) and thecells are incubated for an additional 30 s at 37° C. degrees. After thisincubation, the cells are washed and lysed. The activity of GPR86 inthis cellular extract in the absence or presence of ADP ligand isdetermined by detecting the associated activity of downstream secondmessengers such as cAMP, MAP kinase/ERK phosphorylation and IP₃ asdescribed in examples 3 and 4. GPR86 activity is defined as a two foldor greater increase in ERK phosphorylation or two fold or greaterdecrease in cAMP levels in the absence versus the presence of ADP.Activity is also defined by a two fold or preferably greater change insecond messenger levels in the presence versus the absence of an optimalconcentration of the ligand ADP.

Example 7

Partial Agonist Effect of ATP and 2MeSATP.

The present invention relates, in part, to the activation of GPR86 byADP, or analog or equivalent molecule such as 2MeSADP, ADPβS, or any ofthe ADP analogs taught in U.S. Pat. No. 5,700,786. Accordingly, two ADPanalogs, ATP and 2MeSATP, were tested on AG32 cells transfected with thehuman P2Y13 receptor as describe above. AG32 cells are 1321N1 cellstransfected with Gα16 protein.

AG32 cells were seeded (200,000 cells per dish) on 35 mm (diameter)Petri dishes and labeled for 24 h with 5 μCi/ml [3H]-myoinositol ininositol free DMEM containing 5% fetal calf serum, antibiotics,amphotericin, sodium pyruvate, and 400 μg/ml of G418. Cells wereincubated for 2 h in Krebs-Ringer Hepes (KRH) buffer (124 mM NaCl, 5 mMKCl, 1.25 mM MgSO₄, 1.45 mM CaCl₂, 1.25 mM KH₂PO₄, 25 mM Hepes (pH 7.4)and 8 mM D-glucose). The cells were then incubated in presence of ADP,ATP or 2MeSATP for 30 s. The incubation was stopped by the addition of 1ml of an ice-cold 3% perchloric acid solution. Prior to the stimulation,ATP and 2MeSATP (1 mM solutions) were incubated for 90 min at roomtemperature with 20 units/ml creatine phosphokinase (CPK) and 10 mMcreatine phosphate (CP). The reaction was stopped by addition of 10 mMiodoacetamide.

After treatment with creatinephosphate and creatinephosphokinase, ATPand 2MeSATP reavels to be partial agonists of human P2Y13 receptor. FIG.8 shows the concentration response curve of GPR86 activitationstimulated by the ADP analogs ATP and 2MeSATP. Data is shown as agonistconcentration ploted against [³H]IP₃ counts per minute. ATP and 2MeSATPstimulate GPR86 receptor activity with an EC₅₀ of 4.2±0.8 μM and 1.5±0.2μM, respectively. The data represent the mean±the standard deviation oftriplicate experimental points obtained in one representative experimentof three.

Example 8

Diadenosine Polyphosphates Activity.

The ADP analogs Ap3A, Ap4A, Ap5A and Ap6A were tested on AG32 cellstransfected with the human P2Y13 receptor as describe above.

AG32 cells were seeded (200,000 cells per dish) on 35 mm (diameter)Petri dishes and labeled for 24 h with 5 μCi/ml [³H]-myoinositol ininositol free DMEM containing 5% fetal calf serum, antibiotics,amphotericin, sodium pyruvate, and 400 μg/ml of G418. Cells wereincubated for 2 h in Krebs-Ringer Hepes (KRH) buffer (124 mM NaCl, 5 mMKCl, 1.25 mM MgSO₄, 1.45 mM CaCl₂, 1.25 mM KH₂PO₄, 25 mM Hepes (pH 7.4)and 8 mM D-glucose). The cells were then incubated in presence of twodifferent concentrations (100 nM and 10 μM) of ADP or ApnA (n=3-6) for30 s. The incubation was stopped by the addition of 1 ml of an ice-cold3% perchloric acid solution.

After a 30 s of incubation, the effect of Ap₃A on IP₃ accumulation wasalmost equal to that of ADP (FIG. 9) as measured by production of[³H]IP₃. In contrast, Ap₄A, Ap₅A and Ap₆A were inactive. The datarepresent the mean±the standard deviation of triplicate experimentalpoints obtained in one representative experiment of three.

Example 9

Poly[A] Activity.

Poly[A] and Poly[A].[G] were tested on AG32 cells transfected with thehuman P2Y₁₃ receptor as describe above. The reason of testing Poly[A]and Poly[A].[G] on P2Y₁₃ receptor is the recent evidence that mRNAs canactivate dendritic cells (Weissman et al, 2000, Journal of Immunology,165 : 4710-4717). Thus, these compounds could be used in dendritic cellvaccination therapy as targets of human P2Y13 receptor.

AG32 cells were seeded (200,000 cells per dish) on 35 mm (diameter)Petri dishes and labeled for 24 h with 5 μCi/ml [³H]-myoinositol ininositol free DMEM containing 5% fetal calf serum, antibiotics,amphotericin, sodium pyruvate, and 400 μg/ml of G418. Cells wereincubated for 2 h in Krebs-Ringer Hepes (KRH) buffer (124 mM NaCl, 5 mMKCl, 1.25 mM MgSO₄, 1.45 mM CaCl₂, 1.25 mM KH₂PO₄, 25 mM Hepes (pH 7.4)and 8 mM D-glucose). The cells were then incubated in presence of ADP(as an indicator of optimal stimulation), poly[A] or poly[A].[G] for 30s. The incubation was stopped by the addition of 1 ml of an ice-cold 3%perchloric acid solution. Poly[A] and Poly[A].[G] (1 mM solutions) wereincubated for 90 min at room temperature with 20 units/ml creatinephosphokinase and 10 mM creatine phosphate. The reaction was stopped byaddition of 10 mM iodoacetamide. FIG. 10 shows theconcentration-response curve for activation of human GPR86 with Poly[A]and Poly[A].[G].

After treatment with creatinephosphate and creatinephosphokinase,Poly[A] is still able to activate GPR86, albeit with a lower EC₅₀(205±60 μM), however, Poly[A].[G] is no longer active on human GPR86(data shown in FIG. 10). Concentration of Poly[A] and Poly[A].[G] wereexpressed in AMP equivalent. The data represent the mean±the standarddeviation of triplicate experimental points obtained in onerepresentative experiment of three.

Example 10

Antagonist Activity of Reactive Blue 2, Suramine, PPADS and MRS-2179.

Antagonist were tested on AG32 cells transfected with human GPR86. AG32cells were seeded (200,000 cells per dish) on 35 mm (diameter) Petridishes and labeled for 24 h with 5 μCi/ml [³H]-myoinositol in inositolfree DMEM containing 5% fetal calf serum, antibiotics, amphotericin,sodium pyruvate, and 400 μg/ml of G418. Cells were incubated for 2 h inKrebs-Ringer Hepes (KRH) buffer (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO₄,1.45 mM CaCl₂, 1.25 mM KH₂PO₄, 25 mM Hepes (pH 7.4) and 8 mM D-glucose).Prior to the stimulation, cells were pre-incubated for 10 min inpresence of Reactive Blue 2 (RB-2), Suramine, PPADS or MRS-2179. Thecells were then incubated in presence of ADP 100 nM for 30 s. Theincubation was stopped by the addition of 1 ml of an ice-cold 3%perchloric acid solution. FIG. 11 shows the concentration-response curvefor GPR86 activation by ADP in the presence of the antagonist compounds.The data represent the mean±S.D. of triplicate experimental pointsobtained in one representative experiment of three.

The following table contains the IC50 for the respective compounds.

Potencies of Antagonist in Human P2Y13-AG32 Cells.

Value represent the means±S.D. of three independent experiments.Antagonist IC₅₀ Reactive Blue 2 1.9 ± 0.1 μM Suramine 2.3 ± 0.4 μM PPADS11.7 ± 0.9 μM MRS-2179 >100 μM

Example 11

Screening for Modulators of GPR86 Activity

Candidate modulators of GPR86 can be identified as follows: The assaydescribed in Example 6 provides a premise for screening differentcandidate compounds for ‘modulating’ activity of GPR86. According tothis scenario, GPR86 stably transfected cells are co-incubated with anappropriate concentration of ADP ligand (preferably in the range from 1nM to 1 μM) and different concentrations of an agonist, inverse agonist,antagonist or other candidate modulator compound (preferably in therange from 0.1 nM to 1 μM or more). After incubation at roomtemperature, the cells are washed and lysed. Aliquots of cell extractare then tested in second messenger assays (as described previously inexamples 3, 4 and 6). In this manner, the effect of modulator compoundson GPR86 activity can be measured by determining the activity ofdownstream second messengers in the presence or absence of a candidatemodulator compound under optimal test conditions of ADP ligandconcentration, buffer composition, incubation time and temperature. Theassay can also be performed in a high throughput format (as described inthe kit section) to simultaneously test multiple candidate modulators ata variety of concentrations. GPR86 activity, in the presence of anoptimal concentration of ADP, is determined by detecting any change insecond messenger levels in the presence versus the absence of candidatemodulator compound at a defined concentration.

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing detailed description is providedfor clarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

REFERENCES

-   1. Abbrachio, M. P. and Burnstock, G. (1994) Pharmacol. Ther. 64,    445-475.-   2. Fredholm, B. B. et al. (1997) Trends Pharmacol. Sci. 18, 79-82.-   3. Webb, T. E. et al. (1993) FEBS Lett. 324, 219-225.-   4. Léon, C. et al. (1997) FEBS Lett. 403, 26-30.-   5. Communi, D. et al. (1997) J. Biol. Chem. 272, 31969-31973.-   6. Lustig, K. D. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,    5113-5117.-   7. Parr, C. E. et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91,    3275-3279.-   8. Bogdanov, Y. et al. (1997) J. Biol. Chem. 272, 12583-12590.-   9. Boyer, J. L. et al. (2000) Mol. Pharmacol. 57, 805-810.-   10. Webb, T. E. et al. (1996) Mol. Pharmacol. 50, 258-265.-   11. Chang, K. et al. (1995) J. Biol. Chem. 270, 26152-26158.-   12. Communi, D. et al. (1996) Biochem. Biophys. Res. Commun. 222,    303-308.-   13. Nicholas, R. A. et al. (1996) Mol. Pharmacol. 50, 224-229.-   14. Communi, D. et al. (1995) J. Biol. Chem. 270, 30849-30852.-   15. Nguyen, T. et al. (1995) J. Biol. Chem. 270, 30845-30848.-   16. Webb, T. E. et al. (1996) Biochem. Biophys. Res. Commun. 219,    105-110.-   17. Akbar, G. K. M. et al. (1996) J. Biol. Chem. 271, 18363-18367.-   18. Yokomizo, T. et al. (1997) Nature 387, 620-624.-   19. Li, Q. et al. (1997) Biochem. Biophys. Res. Commun. 236,    455-460.-   20. Janssens, R. et al. (1997) Biochem. Biophys. Res. Commun. 226,    106-112.-   21. Zhang, F. L et al. (2001) J. Biol. Chem. 276 (11), 8608-8615.-   22. Hollopeter, G. et al. (2001) Nature 409, 202-207.-   23. CHAMBERS, J. K. ET AL. (2000) J. BIOL. CHEM. 275 (15),    10767-10771.-   24. Wittenberger, T. et al. (2001) J. Mol. Biol. 307, 799-813.-   25. Communi, D. et al. (1995b). Circ. Res., 76, 191-198.-   26. Brooker, G. et al. (1979) Adv. Cyclic Nucleotide Res. 10, 1-33.-   27. Minamide, L. S. and Bamburg, J. R. (1990) Anal. Biochem. 190,    66-70.-   28. Erb, L. et al. (1995) J. Biol. Chem. 270, 4185-4188.-   29. Baltensperger, K. and Porzig, H. (1997) J. Biol. Chem. 272,    10151-10159.-   30. Eason, M. G. et al. (1992) J. Biol. Chem. 267 (22), 15795-15801.-   31. Chabre, O. et al. (1994) J. Biol. Chem. 269 (8), 5730-5734.-   32. Boyer, J. L. et al. (1993) J. Pharmacol. Exp. Ther. 267,    1140-1146.-   33. Simon, J. et al. (2001) Br. J. Pharmacol. 132, 173-182.-   34. Gudermann et al. (1995) J. Mol. Med. 73, 51-63.

1-45. (canceled)
 46. A method of detecting the dysregulation of GPR86activity, said method comprising: a) contacting a sample with a ligandspecific for a GPR86 polypeptide; and b) detecting an activity of saidGPR86 polypeptide in said sample, wherein a difference in said activityrelative to a standard is indicative of dysregulated GPR86 activity. 47.The method of claim 46, wherein said ligand is an antibody or antigenbinding fragment thereof.
 48. The method of claim 46, wherein saidligand is selected from the group consisting of ADP, 2MeSADP, ADPbetaSand an ADP analog.
 49. The method of claim 46, wherein said ligand isdetectably labeled.
 50. The method of claim 49, wherein said ligand isdetectably labeled with a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, andan affinity tag.
 51. The method of claim 46, wherein said activity isbinding of said ligand to said GRP86 polypeptide.
 52. The method ofclaim 51, wherein said ligand is an antibody or an antigen bindingfragment thereof.
 53. The method of claim 51, wherein said ligand isselected from the group consisting of ADP, 2MeSADP, ADPbetaS and an ADPanalog.
 54. The method of claim 46, wherein said activity is a signalingactivity of said GPR86 polypeptide.
 55. The method of claim 54, whereinsaid step of detecting said signaling activity of said GPR86 polypeptidecomprises detecting a change in the level of a second messenger.
 56. Themethod of claim 54, wherein the step of detecting the signaling activitycomprises measurement of guanine nucleotide binding or exchange,adenylate cyclase activity, cAMP, protein kinase C activity,phosphatidylinosotol breakdown, diacylglycerol, inositol triphosphate,intracellular calcium, arachinoid acid concentration, MAP kinaseactivity, tyrosine kinase activity, or reporter gene expression.
 57. Themethod of claim 46, wherein said sample is a tissue sample.
 58. Themethod of claim 46, wherein said sample is a cell membrane preparation.59. The method of claim 46, wherein said sample is a cell preparation.60. The method of claim 46, wherein said standard comprises apolypeptide having the amino acid sequence of SEQ ID NO:1.